14_1 Cultivo Cel Animales Gibco Catalogue Edition 49

Important Licensing Information: The products in this catalogue may covered by one or more Limited Use Label Licenses (see the Invitrogen website www.invitrogen.com). By use of these products, you accept the terms and conditions of all applicable Limited Use Label Licenses. All products are for research use only, and are not intended for human or animal diagnostics, therapeutic, commercial, or other clinical uses unless otherwise specifically indicated in the applicable product Limited Use Label License(s).

gibco® cell culture catalogue

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IntroductionWhen reliable results and confidence in your work are critical to your success, trust gibco® products and services. Quality is built into every gibco® product, from the finest raw materials to precise formulations and rigorous final product testing. Gibco® products are consistent over time, lot to lot and bottle to bottle. To you, that means greater reliability, greater consistency, greater control, and ultimately, greater confidence in your results.

Comprehensive range of products provides what you need, when you need it ■

Lot to lot, bottle to bottle, gibco® products are reliably consistent over time ■

Gibco® quality goes into every product, from the highest-quality raw materials to our rigorous final product testing ■

Manufactured in cGMP facilities, certified to FDA Regulations 21 CFR Part 820 and ISO 13485:2003 ■

to meet stringent quality standardsNew and innovative products designed to simplify your workflow and improve your Cells health and performance ■

Advancing the robustness of your experiments through our leadership in enabling ■

Advanced cell systems for research ■

Our award-winning technical service group is your resource for products, techniques, troubleshooting and applications ■

Better results begin with better cell culture. To learn more about the gibco® collection of Products and services, visit www.invitrogen.com/gibco

Resources web linksThe gibco®cell culture basics handbook:www.invitrogen.com/cellculturebasics

cell culture protocol: www.invitrogen.com/protocols

Information on mammalian and insect cell culture cell type specific protocols and additional cell culture products: www.invitrogen.com/cellculture

Technical Support, Safety data sheets (SDS) are available at: www.invitrogen.com/sds

Certificate of Analysis provides detailed quality control and products qualification information for each product. Certificates of Analysis are available on our website www.invitrogen.com/support, search for the certificate of analysis by product lot number which is printed on the box.

Media Selection tools:http://igene.invitrogen.com/products/selector/pscshttp://igene.invitrogen.com/products/selector/media

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Introduction

Cell Culture ProductsThis information applies to the following cell culture product chapters, with the exception of Chapter 8, Animal Sera.

The products listed in the following chapters are “for research use and further cell culture manufacturing only,” except where otherwise denoted. These products are not for diagnostic use. The safety and efficacy for these products in diagnostic or other clinical uses have not been established. In appropriate circumstances these products may be used as raw material components in further manufacturing applications.

Products listed “for in vitro diagnostic use” are medical devices subject to the requirements of the Code of Federal Regulations Title 21, Part 820: Quality System Regulation. These products may also be used for research applications, and in appropriate circumstances, as raw material components in further manufacturing applications. It is the end user ’s responsibility to qualify these products for their specific appli cation. They have not been qualified nor are they intended for any animal or human therapeutic uses.

Custom Cell Culture ProductsWe have the technical and manufacturing capabilities to produce a wide variety of customized cell culture media and reagents. From slight formulation modifications to complicated engineered-to-order products, custom products are available in both standard and highly specialized packaging configurations. Wherever possible, we can formulate custom media with animal origin–free components and offer developmental support to help you re-engineer formulations to meet regulatory and performance goals. Gibco® is the only brand that offers four distinct formats for media: ready-to-use (1X) liquid media, dry powder media (DPM), liquid medium concentrates (LMCs), and Advanced Granulation Technology™ (AGT™).

Whether you need media for cell culture or buffers for purification, submit your requests to our Custom Products Services group for timely evaluation and production of the formulation and delivery system that best fits your needs.

Custom liquid medium concentratesUnique features of liquid medium concentrates include improved nutrient solubility, superior correlation with theoretical nutrient potency, and glutamine stabilization.

Advanced Granulation Technology™ (AGT™)AGT™ is a dry form of cell culture media in a new granular format that is optimally suited to research and industrial-scale production applications. By applying the well-characterized pharmaceutical manufacturing technology of fluid-bed granulation to cell culture media, we can now offer modern, highly complex formulations in an easy-to-use format.AGT™ media are complete and pH/OSMO adjusted. The essential benefit of a complete AGT™ medium is that it provides one raw material for the user and can decrease costs and time involved in raw material planning, procurement, and testing. Medium preparation is also simplified because

an AGT™ medium is complete and does not require pH/OSMO adjustment. Additionally, because the granules wet instantly, providing easier reconstitution, overall medium preparation time is reduced.

Containers and closuresThe following flexible plastic bag packages are available for liquid media and most reagents: 1 L, 5 L, 10 L, 20 L, 100 L, and 200 L. Stock config u ra tions of these units have been designed for general utility and convenience, but the customization of these bags to meet your specific needs is available. More information about our line of custom packaging can be obtained at www.invitrogn.com/synergy, through your Account Manager, or by contacting our Custom Products Services group.

Production and PreparationLiquid cell culture productsWater preparation. The water used in liquid cell culture products is distilled water meeting all USP Monograph test requirements for Water For Injection. The system includes water pretreatment followed by distillation.

The pure distillate is collected in storage tanks for holding until distribution via a constantly recirculating loop to points of use. The water is cooled to formulation temperature by a heat exchanger. All piping in the still, storage, and distribution systems is constructed of sanitary 316L stainless steel. The system is controlled automatically by a microprocess controller employing state-of-the-art sensing and fail-safe systems.

Continuous quality assurance testing for microbial bioburden, endotoxin, and Water For Injection USP monograph requirements ensures that all specifications are met.Addition of chemicals. All raw material components used in our liquid medium formulations undergo stringent quality control tests, such as for identity and purity, to provide assurance that our media meet their final product quality control specifications. The biochemicals used in our formulations are USP, ACS, FCC, or other compendial grade. When no published standard is available, we set internal specifications (gibco® grade) to ensure optimal product performance.

Chemicals of confirmed high quality are weighed accurately and confirmed using a state-of-the-art computerized weighing system. The chemicals are then solubilized in distilled water in calibrated formulation tanks. In each batch preparation, mixing is maintained to ensure homogeneity.Membrane filtration. gibco® cell culture media are prepared by an aseptic filling process for which each step has been validated to ensure the production of a product meeting the industry-standard sterility assurance level of 10–3 (i.e., product demonstrates a contami nation level of no more than 1 of 1,000 units during the manufacturing process). The highest level of sterility assurance (equal to or greater than 10–6) cannot be achieved without terminal sterilization, which can be harmful to the performance of some cell culture products. Key points of control include validated sterilization cycles for all material with product contact, routine medium fills employing bacterio logical media, a comprehensive environmental monitoring program, validated cleaning procedures, and a validated



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final filter integrity testing program. Additionally, filtration and dispensing are performed within positive-pressure, HEPA-filtered, environmentally controlled rooms.

The filled containers are labeled and maintained under quarantine at their required temperatures until quality control tests are complete and the product meets our final quality control release specifications. Prior to release, all production records and test results undergo a final review by our Quality Assurance Team.

Powdered Cell Culture ProductsAll raw material components used in our powdered medium formu lations undergo stringent quality control tests, such as for identity and purity, to provide assurance that our media meet their final product quality control specifications. The biochemicals used in our formulations are USP, ACS, FCC, or other compendial grade. When no published standard is available we set internal specifications (gibco® grade) to ensure optimal product performance.

Quality SystemsOur quality systems and processes ensure our success in meeting your needs.

ISO 13485 certified facility at Inchinnan, Scotland ■

We are audited by major biopharma and diagnostic companies ■

We employ Six Sigma and Lean Manufacturing to ensure top quality ■

and lower costsOur stringent management controls, process controls, and built-in

process improvement steps ensure we meet the quality standards that you expect. We aim to meet quality and regulatory standards and to exceed customer expectations in all that we do. At Invitrogen, we welcome customer and supplier audits as a way to ensure our quality standards meet the expectations and ever-changing needs of our customers.

Quality ControlLiquid mediaTo ensure that our validated systems and controls continue to be effective, the quality of our liquid media is confirmed by testing samples from each production batch.Chemical checks. Osmolality and pH are measured to verify compliance with product specifications.Analytical component testing. Some media products and supplements are tested using analytical techniques like HPLC to verify that contents of the media formulation have been delivered within acceptable limits to assure product performance.Microbiological tests. For liquid media, the absence of bacterial and fungal contamination is confirmed using the method referenced in the current edition of the U.S. Pharmacopeia sterility test monograph. Also, products of animal origin are tested for the absence of mycoplasmas using the large-volume inoculation method of Barile and Kern.1 For details of these tests, see Chapter 4, “Microbiological Tests.”Growth performance and cytotoxicity tests. Many liquid media are tested for the absence of cytotoxicity and for growth promotion in a suspension culture assay. A rapidly growing murine myeloma cell line, Sp2/0-Ag14, is

inoculated at approximately 5 × 104 cells/ml and the logarithmic growth rate monitored relative to parallel cultures grown in a standardized reference medium. Specialized culture media are performance tested on appro priate cell lines compared to recognized reference standards.Endotoxin tests. Endotoxin values are measured with the Limulus Amebocyte Lysate (LAL) test. Media with the lowest level of endotoxin attainable in the industry today are routinely available.Stability testing program. The expiration date shown on every in vitro diagnostic (IVD)-labeled product is an indication of how long the product will deliver effective performance. Our stability program establishes and verifies product expiration dates. Product groups are monitored at periodic intervals to determine the length of time that the product will deliver acceptable performance under recommended storage conditions.

Powdered mediaThe quality of powdered media is confirmed by evaluating samples from each batch as required by specifications. Samples are prepared in 1X liquid form and membrane filtered, and testing may include:

Growth promotion/toxicity ■

Solubility ■

Osmolality ■

pH ■

The values for these parameters are determined using the test conditions described for liquid media. All powdered media are produced without sodium bicarbonate to increase stability.

Product datingPlease refer to product labels for expiration dates of products.

Storage recommendationsFor sustained optimal performance, store cell culture products under the conditions recommended on the labels, and minimize exposure of liquid media to all light sources under both storage and usage conditions.2–5 Keep powder formulations in the dark and dry.

Certificates of AnalysisCertificates of Analysis for all cell culture products are available upon request. These documents supplement our standard label data and published specifications with the results of testing performed on all gibco® cell culture liquid media and reagents. Also provided is important information about intended product use in keeping with published FDA guidelines.

References1. Barile, M.F. and Kern, J. (1971) Proc. Soc. Exp. Biol. Med. 138, 432.2. Wang, R.J. (1976) In Vitro 12, 19.3. Wang, R.J. and Nixon, B.T. (1978) In Vitro 14, 775.4. Spierenburg, G.T., Oerlemans, F.T., VanLaarhoven, J.P., and de Bruyn, C.H. (1984)

Cancer Res. 44, 2253.5. Parshad, R., Taylor, W.G., Sanford, K.K., Camalier, R.F., Gantt, R., and Tarone, R.E.

(1980) Mutat. Res. 73, 115.

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Ordering and Technical SupportFor your convenience, you may place your order by the method that is best for you: choose to order online, by email, phone, mail, or fax. Our highly efficient Customer Service representatives will process your order quickly and accurately.

Order OnlineYou have two options for placing orders electronically:

Place your order online 24 hours a day, 7 days a week, ■

at our website: www.invitrogen.comEmail your order: For specific country email addresses, ■

refer to the back of the catalogue

Order by Phone, Mail, or FaxWhen you order, please include the following information:1. Institution or company customer number2. Billing address3. Shipping address4. Valid Purchase Order number5. Name of purchasing agent and department6. Name and phone number of end user7. Catalogue number and description of product8. Quantity and size of product

EUROPEAN HEADQUARTERSLife Technologies102 Fountain CrescentPaisley, Scotland PA49REUnited Kingdomwww.invitrogen.com

Technical/Customer SupportOur Technical Support lines are open from 6:00 a.m. to 5:00 p.m. Pacific Standard Time. Customer Service represen tatives are here to assist you with ordering from 5:00 a.m. to 5:00 p.m. Pacific Standard Time.Contact us from anywhere in the US:

Technical Support ■

email technical enquiries . . [email protected] general enquiries . . . . [email protected] . . . . . . . . . . . . . . . . . . . . .www.invitrogen.com/support

Customer Service ■

email . . . . . . . . . . . . . . . . . . . . . .For specific country email addresses, refer to the back of the catalogue

Need order status or want to track a shipment? Have a question or need help with an order? Go to www.invitrogen.com/support and take advantage of our convenient new customer support tools, any time of the day or night.

International OrdersInternational customers should order directly from the nearest authorized distributor of Invitrogen products. For the location of the nearest distributor, refer to the distributor address list on the inside back cover of this catalogue or at www.invitrogen.com. Where no distributor is designated, orders may be placed with Invitrogen Corporation directly. Shipments outside the continental United States are sent air prepaid. Direct international orders shall require prepayment, payable in U.S. funds via bank wire transfer, Visa®, Mastercard®, American Express®, or the American Express® Corporate Purchasing Card. Freight charges will be collected whenever possible.



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Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . i

Resources web links . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . i

Cell Culture Products. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ii

Ordering and Technical Support. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . iv

Chapter 1 PSCS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2

Gibco® Cell Therapy Systems (CTS™) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2

Gibco® CTS™ Immunotherapy products . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3

Gibco® CTS™ products for stem cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4

CELLstart™ Cell Attachment/Expansion Substrate. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4

Geltrex™ Reduced Growth Factor Basement Membrane Matrix . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5

Collagen 1 Bovine and Rat Tail Collagen I . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5

AlgiMatrix® 3D Culture System. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6

Natural Mouse Laminin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6

Chapter 2 Growth factors, Cytokines and Chemokines with high bioactivity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9

Growth factors, Cytokines and Chemokines with high bioactivity. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10

Gibco® Recombinant human FGF-basic . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12

Gibco® LIF Recombinant . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13new Gibco® Recombinant Growth Factors for use in stem cell applications . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14

Human proteins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15

Rat proteins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 18

Swine proteins. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19

Publication References. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20

Chapter 3 Basal Media . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 23

Reduced-Serum Media at a Glance . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25

GlutaMAX™ Media at a Glance . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 26

HEPES Media at a Glance . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 27

Phenol Red–free Media at a Glance. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 28

Basal Medium Eagle . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 28

BGJb Medium (Fitton-Jackson Modification) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 28

Brinster’s BMOC-3 Medium . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 29

Cryopreservation Medium . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 29

CMRL Medium. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 29

CO2-Independent Medium . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 29

Dulbecco’s Modified Eagle Media (DMEM) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 30

F-10 Nutrient Mixtures . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 34


Glasgow Minimum Essential Medium . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35

Grace’s Insect Cell Culture Media . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35

Improved MEM Zn++ . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 36

IPL-41 Insect Medium . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 36

Iscove’s Modified Dulbecco’s Media . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 36

Leibovitz’s L-15 Media . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 37

McCoy’s 5A Medium (Modified) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 37

MCDB 131 Medium. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 37

Media 199 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 38

Minimum Essential Media (MEM) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 39

Advanced MEM (Minimum Essential Medium). . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 40

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Minimum Essential Medium (MEM) with Earle’s Salts . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 40

Minimum Essential Medium (MEM) with Hanks’ Salts. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 42

Modified Eagle Media . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 42

Medium NCTC-109 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 42



Opti-MEM® I Reduced-Serum Media . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 43

Lipofectamine™ 2000 Transfection Reagent . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 43

RPMI Media 1640 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 44

Schneider’s Drosophila Medium. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 46

Waymouth’s MB 752/1 Medium . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 46

Williams’ Medium E . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 46

Media Preparation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 47

E-Z Hold™ Plastic Bottles . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 49

Stak Pack™ Accessories . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 49

Media Formulations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 50

Chapter 4 Serum-free and Specialty Media and Reagents . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 83

Summary of Serum-Free, Protein-free, and Chemically Defined Media. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 85

Summary of Specialty Media . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 85

Serum-free Media and Reagents . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 86

Specialty Reagents . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 87

3D Matrices and Scaffolds. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 88

Expression Products . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 89

Cellular Studies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 109

Stem Cell Research Products . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 117

Cytogenetics Products. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 126

Overview of Transfection, Selection, and Protein Delivery Methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 131

Transfection Reagents . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 132

Chapter 5 Balanced Salt Solutions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 137

Balanced Salt Solutions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 138

Dulbecco’s Phosphate-Buffered Salines . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 138

Earle’s Balanced Salt Solution. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 139

Hanks’ Balanced Salt Solutions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 140

Phosphate-Buffered Saline (PBS) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .141

Balanced Salt Solutions Preparation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 142

Balanced Salt Solutions Formulations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 143

Chapter 6 Standard Cell Culture Reagents . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 147

Cell Culture Reagents . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 148new ACK Lysing Buffer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .149

Amino Acids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 149

Antibiotics and Antimycotics. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 150

Antifoaming and Defoaming Reagents . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 156

Biological Buffers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 157

Cell Dissociation Products. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 158

Distilled Water . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 161new WFI for Cell Culture (Water for Injection) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 161

Attachment Factors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .162

Growth Supplements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .163



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Hybridoma Reagents . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 166

Insect Medium Supplements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .167

Mycoplasma Detection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 167

Other Cell Culture Reagents . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 168

Cell Culture Reagent Formulations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 169new Presto Blue™. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 170

Chapter 7 Eukaryotic Cell Lines . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .173

Insect Cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 174

Mammalian Cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 177

Cell Line Development Kits . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 180

new OptiCHO™ Express Kit . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .180

new OptiCHO™ Antibody Express Kit. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 181

new Gibco® Hepatocytes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .182

Chapter 8 Animal Sera . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 185

Fetal Bovine Sera (FBS) and other Sera for Cell Culture . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 186

FBS Selection Guide. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 187

Fetal Bovine Sera (FBS). . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 187

Specialty FBS Selection Guide. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 189

Other Sera Selection Guide . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 190

Newborn Calf Sera . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 191

Bovine Sera . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 191

Horse Sera . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .192

Other Sera . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .192

FBS Quality Control Test Comparison. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 193

Other Sera Quality Control Test Comparison . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 194

Final Product Quality Control. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 196

Performance Tests: Fetal Bovine Sera (FBS) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 197

Special Proccessing of Sera . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 200

Appendix I . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .203

Gibco® Universal Bags . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 204new Lab Armortm Beads . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .208

PD-Directtm Bioprocess Services . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 209

Cell Culture basics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .210new Supply Centre by Life Technologiestm . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 213

Formulas and Formula Weights for Commonly Used Medium Components . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 216

Troubleshooting: Cell Culture . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 218

Useful Numbers for Cell Culture . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .219

Buffer Systems for Cell Culture . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 219

Specialty Media and Applications . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 219

Serum-free Media and Applications . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 220

Adaptation of Cell Cultures to a Serum-free Medium. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 224

Adaptation of Cell Cultures to an Alternative Serum . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 225

Adaptation of Cells from Monolayer to Suspension Culture . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 225

Stability of GlutaMAX™-I vs. L-Glutamine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 226

Cryopreservation of Mammalian Cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 227

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Thawing of Cryopreserved Cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 227

Dissociation of Cells from Primary Tissue . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 228

Dissociation of Cells from Culture Vessels . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 229

Decontaminating Cultures with Antibiotics and Antimycotics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 231

Use of Antibiotics and Antimycotics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 231

Conversion Factors, Units, and Abbreviations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 232

Genetic Code and Amino Acid Abbreviations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 233

Appendix II . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .235

Terms and Conditions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 236

Trademark Information . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .238

Index . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .239

Alphabetical Index . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 240

Catalogue Number Index . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 246

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Gibco® Cell Therapy Systems (CTS™) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2

Gibco® CTS™ Immunotherapy products . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3

AIM V® Medium CTS™—Therapeutic-grade, serum-free cell expansion medium . . . . . . . . . . . . . 3

OpTmizer™ CTS™ T-Cell Expansion SFM . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3

CTS™ cytokines and growth factors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3

Gibco® CTS™ products for stem cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4

CELLstart™ CTS™ Cell Attachment/Expansion Substrate . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4

Gibco® Extracellular Matrices . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5

Geltrex™ Reduced Growth Factor Basement Membrane Matrix . . . . . . . . . . . . . . . . . . . . . . . . 5

Collagen 1 Bovine and Rat Tail Collagen 1 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5

AlgiMatrix™ 3D Culture System . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6

Natural Mouse Laminin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6

Chapter 1 PSCS

Chapter 1 PSCS

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Gibco® Cell Therapy Systems (CTS™)As you move your translational stem cell, tissue engineering, and immunotherapy research toward the clinic, high-quality products and proper documentation are essential to “getting it right the first time”. The gibco® Cell Therapy Systems (CTS™) brand offers a broad array of high-quality products designed for use in cell therapy applications, including media, reagents, growth factors, enzymes, selection beads, and devices which are manufactured in compliance with 21 CFR Part 820 Quality System Regulation and/or are certified to ISO 13485 and ISO 9001.

CTS™ products are supplied with unified documentation such as— Certificates of Analysis ■

Certificates of Origin ■

Access to authorization letters for our FDA Drug Master File ■

Effective cell therapies require quality raw materials, and that is what you can expect from CTS™. The CTS™ product line will help reduce your burden in qualifying reagents as you prepare to transition your research into clinical applications. The FDA requires manufacturers of cell therapy products to qualify raw materials (reagents, media, growth factors, etc.) used in cell therapy manufacturing processes. Relevant CTS™ products are mycoplasma-, endotoxin-, and sterility-tested to meet these regulations. In addition, raw materials are USP/EP grade, or an equivalent grade when available. Products labeled RUO or not manufactured under GMPs can create a challenge for the FDA, as performance standards or quality attri-butes may not have been established. To mitigate these issues, we have established Device Master Files (DMFs) for many of our CTS™ products to support product development activities.

For more information on CTS™ products, visit www.invitrogen.com/gibcocts or email us at [email protected].

gibco® cell culture cataloguePSCS

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3www.invitrogen.com

Gibco® CTS™ Immunotherapy products Whether you’re developing cell-based vaccines, gene-modified T cells, or other immune-based cell therapy strategies, gibco® CTS™ delivers tools and reagents to support all aspects of your work—from bench to bedside and beyond—into commercial applications. Gibco® CTS™ provides specialized products to grow, isolate, and expand T cells, dendritic

AIM V® Medium CTS™—Therapeutic-grade, serum-free cell expansion mediumGibco® AIM V® Medium CTS™ (Therapeutic Grade) is the first commercially available defined, serum-free formulation for proliferation and/or manipulation of T cells and dendritic cells and is manufactured in compliance with cGMP. AIM V Medium CTS™ is an FDA 510(k)–cleared device which is intended for human ex vivo tissue and cell culture processing applications. CAUTION: When used as a medical device, federal law restricts this device to sale by or on the order of a physician.

OpTmizer™ CTS™ T-Cell Expansion SFMGibco® OpTmizer™ CTS™ T Cell Expansion SFM has been developed for the growth and expansion of human T lymphocytes. OpTmizer™ CTS™ T-Cell Expansion Medium is a complete serum-free, xeno-free 1X medium consisting of OpTmizer™ T Cell Expansion Basal Medium and the OpTmizer™ T-Cell Expansion Supplement. For research use or manufacturing of cell-, gene-, or tissue-based products. CAUTION: Not intended for direct administration into humans or animals.

CTS™ cytokines and growth factorsWith highly active, highly pure gibco® CTS ™ cytokines and growth factors, you can culture, expand, and differentiate cell populations with more confidence in your results. Gibco® CTS™ cytokines and growth factors are manufactured under ISO13485 quality standards, and are validated with gibco® CTS™ media, further reducing variability to enable reliable results. For research use or non-commercial manufacturing of cell-based products for clinical research.CAUTION: Not intended for direct administration into humans or animals.

Ordering InformationProduct Quantity Cat. no.

AIM VR Medium CTS™ 1,000 mL 087-0112DK

AIM VR Medium CTS™ 10 L 087-0112BK

Dynabeads® CD3/CD28 CTS™ 10 mL 402-03D

DynaMag™ CTS™ each 12102

OpTmizer™ CTS™ T-Cell Expansion SFM 1 L A10485-01

Interleukin 4 CTS™ (IL-4) Recombinant Human 1 mg CTP0043

Interleukin 4 CTS™ (IL-4) Recombinant Human 100 μg CTP0041

GM-CSF CTS™ Recombinant Human 1 mg CTP2013

GM-CSF CTS™ Recombinant Human 100 μg CTP2011

TNF-α CTS™ Recombinant Human 1 mg CTP3013

TNF-α CTS™ Recombinant Human 100 μg CTP3011

Interleukin 7 CTS™ (IL-7) Recombinant Human 1 mg CTP0073


Interleukin 2 CTS™ (IL-2) Recombinant Human 1mg CTP0023


DPBS CTS™ (without Calcium Chloride, 1 L A12856-01

without Magnesium Chloride)

DPBS CTS™ (with Calcium Chloride, 1 L A12858-01

with Magnesium Chloride)

cells, and other cell types. These products are designed to facilitate the development of cell-based vaccines, gene-modified T cells, or other immunity-based cell therapy strategies and to ease your transition into the clinic.

Chapter 1 PSCS

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Gibco® CTS™ products for stem cellsRegardless of the type and source of your stem cells, gibco® CTS™ provides tools to help you isolate, expand, differentiate, and characterize your stem cells. Enter the clinic knowing that your tailored solutions will meet your regulatory and quality requirements.

CELLstart™ CTS™CELLstart™ CTS™ Cell Attachment/ Expansion SubstrateCELLstart™ substrate contains only components of human origin (xenofree) and supports human embryonic stem cell (hESC) and induced pluripotent stem cell (iPSC) attachment, expansion of undifferentiated colonies, maintenance of pluripotency, and differentiation capability in serum-free medium without the need for feeder cells. CELLstart™ substrate can also be used with mesenchymal and neural stem cells, and for the attachment of human feeder cells (e.g., foreskin fibroblasts).


CELLstart™ CTS™ 2 mL A10142-01

DPBS CTS™ (without Calcium Chloride,

without Magnesium Chloride) 1 L A12856-01

DPBS CTS™ (with Calcium Chloride,

with Magnesium Chloride) 1 L A12858-01

GlutaMAX™-I CTS™ 100 mL A12860-01

KnockOut™ SR XenoFree CTS™ Kit 100 mL A1099201

KnockOut™ SR XenoFree CTS™ 100 mL 12618-012

KnockOut™ SR XenoFree CTS™ Kit 500 mL A1099202

KnockOut™ SR XenoFree CTS™ 500 mL 12618-013

KnockOut™ D-MEM CTS™ 500 mL A12861-01

TrypLE™ Select CTS™ 100 mL A12859-01

Interleukin 6 CTS™ Recombinant Human 100ug CTP0061

Interleukin 6 CTS™ Recombinant Human 1mg CTP0063

FGF basic full length CTS™ Recombinant Human 100ug CTP0261

FGF basic full length CTS™ Recombinant Human 1mg CTP0263

Stem Cell Factor (SCF) CTS™ Recombinant Human 100ug CTP2111

Stem Cell Factor (SCF) CTS™ Recombinant Human 1mg CTP2113

TGF beta 1 CTS™ Recombinant Human 100ug CTP9211

TGF beta 1 CTS™ Recombinant Human 1mg CTP9213

Flt-3 Ligand CTS™ Recombinant Human 100ug CTP9411

Flt-3 Ligand CTS™ Recombinant Human 1mg CTP9413

For Research Use or Non-Commercial Manufacturing of Cell-Based Products for Clinical Research.CAUTION: Not intended for direct administration into human or animals.


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Geltrex™ Reduced Growth Factor Basement Membrane Matrix

Geltrex™ matrix is a soluble form of reduced growth factor (RGF) ■

basement membrane extract (BME) purified from continuous sheets of specialized extracellular matrix that form an interface between Engelbreth-Holm-Swarm (EHS) tumor cells. The major components of Geltrex™ matrix include laminin, collagen ■

IV, entactin, and heparin sulfate proteoglycan. Geltrex™ reduced growth factor basement membrane matrix ■

supports cells and cell layers, and plays an essential role in tissue organization that affects cell adhesion, migration, proliferation, and differentiation.Geltrex™ matrix enables, on average, a 2-fold reduction in several ■

growth factors.


Geltrex™ Reduced Growth Factor 1 mL 12760013Basement Membrane Matrix

Geltrex™ Reduced Growth Factor 5 mL 12760021Basement Membrane Matrix

Geltrex™ LDEV Free Reduced Growth Factor 5 mL A1134301Basement Membrane Extract

Geltrex™ hESC-Qualified Reduced Growth Factor Basement Membrane Matrix 1 mL A1048001

Geltrex™ hESC-Qualified Reduced Growth Factor Basement Membrane Matrix 5 mL A1048002

Related products PageHuMEC-SFM . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .114KnockOut™ Serum Replacement . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .120Phosphate-Buffered Saline (PBS) 7.2 (1X) . . . . . . . . . . . . . . . . . . . . . . . . . . . .141TrypLE™ Express Stable Trypsin Replacement Enzyme, without phenol red . . . . . . . . . . . . . . . . . . . . . . . . . .160Versene 1:5,000 (1X) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .161Visit us at www.invitrogen.com/geltrex to learn more about the Geltrex™ 3D Culture System.

Bovine and Rat Tail Collagen ICollagen I is a heterotrimeric extracellular matrix protein found in skin and other connective tissues. Gibco® Bovine and Rat Tail Collagen I for cell culture are high-quality forms of collagen widely used in 3D applications and as coating solutions.

Rat Tail Collagen I is provided in liquid form as well as precoated on ■

plates in 6-well, 24-well, and 96-well formats. Rat Tail Collagen I is the most common fibrillar collagen (90%) found in skin, bone, tendons, and other connective tissues. The liquid form of Rat Tail Collagen I is supplied at 5 mg/mL in a 20 mL vial, providing the flexibility to dilute it to lower concentrations.Bovine Collagen I is a naturally occurring extracellular matrix found ■

in tendons and other connective tissues. It plays a key role in cell growth, differentiation, attachment, and migration. This solution is supplied at 5 mg⁄mL in a 10 mL vial, providing the flexibility to dilute it to lower concentrations.


Collagen I, Bovine 10 mL A1064401

Collagen I, Rat Tail 20 mL A1048301

Coated Plates, Collagen I, Rat Tail 5 x 6-well plates A1142801

Coated Plates, Collagen I, Rat Tail 5 x 24-well plates A1142802

Coated Plates, Collagen I, Rat Tail 5 x 96 well plates A1142803

For Research Use Only. Not intended for animal or human therapeutic or diagnostic use.

Gibco® Extracellular Matrices

Chapter 1 PSCS

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AlgiMatrix® 3D Culture SystemThe AlgiMatrix® 3D Culture System is a user-friendly, non–animal- ■

derived bioscaffold for three-dimensional (3D) cell culture. The AlgiMatrix® sponge is formulated using pharmaceutical-grade raw material from brown seaweed. The AlgiMatrix® 3D Culture System creates a cell culture model system ■

that more closely reflects normal cell morphology and behavior, to help meet researchers’ requirements for 3D cell culture in fields such as toxicology, drug development, cancer and stem cell research, development and morphogenesis, tissue and organ engineering, heart disease, and diabetes.The AlgiMatrix® system is available in 6-well, 24-well, and 96-well ■

formats that fit well into the workflows of both low- and high-throughput users. Cells can be inoculated directly into the sterile microtiter plates preloaded with lyophilized alginate sponge, with no pretreatment or preparation required.

Recommended storage conditions: room temperature.


AlgiMatrix™ 3D Culture System, 1 x 96-well plate 12684015in 96-well plate

AlgiMatrix™ 3D Culture System, 5 x 96-well plates 12684031in 96-well plate

AlgiMatrix™ 3D Culture System 1 x 24-well plate 12684023

AlgiMatrix™ 3D Culture System 4 x 24-well plates 12684049

AlgiMatrix™ 3D Culture System, 1 x 6-well plate A10982016-well kit

AlgiMatrix™ 3D Culture System, 4 x 6-well plates A10982026-well kit

AlgiMatrix™ Firming Buffer 50 mL A1091501

AlgiMatrix™ Dissolving Buffer 50 mL A1134001


Related products PageDefined Keratinocyte-SFM System . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .110Hepatozyme-SFM . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .113KnockOut™ Serum Replacement . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .120Phosphate-Buffered Saline (PBS) 7.2 (1X) . . . . . . . . . . . . . . . . . . . . . . . . . . . .141TrypLE™ Express Stable TrypsinVersene 1:5,000 (1X) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .160Visit www.invitrogen.com/3D-CellCulture to learn more about the AlgiMatrix™ 3D Culture System and related products for 3D cell culture.

Natural Mouse LamininNatural Mouse Laminin is isolated from Engelbreth-Holm-Swarm (EHS) sarcoma cells and is the major glycoprotein component of basement membranes. Among the functions it modulates are cell attachment, cell spreading, cell growth and motility, promotion of epithelial differentiation, modification of leukocyte function, and stimulation of neurite outgrowth.


Natural Mouse Laminin 1 mg 23017-015



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Notes:

Chapter 1 PSCS

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Notes:

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Growth factors, Cytokines and Chemokines with high bioactivity. . . . . . . . . . . . . . . . . . . . . . . . . 10

Gibco® Recombinant human FGF-basic . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12

Gibco® LIF Recombinant . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13

new Gibco® Recombinant Growth Factors for use in stem cell applications . . . . . . . . . . . . . . . . . 14

Thrombopoietin (TPO) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14

BMP-4 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14

BMP-7 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14

Activin A . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14

IL-17F . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15

Human proteins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15

Rat proteins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 18

Swine proteins. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19

Publication References. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20

Growth factors, Cytokines andChapter 2 Chemokines with high bioactivity

Chapter 2 Growth factors, Cytokines, and Chemokines with high bioactivity

10


Proven Compatibilitygibco® recombinant proteins have proven compatibility with gibco® media. gibco® recombinant proteins are bioassayed with gibco® media. You can be confident that your recombinant protein is compatible with the media you trust.

High Biological Activitygibco® recombinant proteins have biological activity that meets or exceeds industry standards. Higher activity provides more results with less protein.

High Puritygibco® recombinant proteins are highly purified. High purity means no interference from other proteins or contaminant. When working with gibco® recombinant proteins you get results you can trust.

How are your cytokines, chemokines, and growth factors manufactured?

High QualityAll gibco® recombinant proteins from our Camarillo, CA, and Frederick, MD, sites are manufactured under ISO 13485 quality standards. These standards require that all proteins deliver high bioactivity, low endotoxin, and consistent performance.

Multiple Packaging SizesMany gibco® recombinant proteins are available in multiple package sizes to meet your application needs. Buffer formulations and package sizes may be customized upon request. For more information, please contact Custom Services at [email protected] or by phone at 800 955 6288.

Growth factors, Cytokines and Chemokines with high bioactivity

Process

Result

Expression• Gene cloned from cDNA• Sequence verified• Expression system chosen

Expressed in a host system that yields a biologically active protein

Purification and refolding• Purification by chromatography• Refolding of proteins expressed

in bacterial system

Purified to produce a highly pure, homogenous solution of active protein

Formulation and lyophilization• Carrier-free• Sterile-filtered prior to

lyophization

formulated and lyophilized to ensure maximum stability

➝ ➝

gibco® cell culture catalogueGrowth factors, Cytokines and Chem

okines with high bioactivity2


11www.invitrogen.com

How is the quality of your growth factors ensured?To ensure gibco® growth factors are of the highest quality, each protein is analyzed for purity along with structural homogeneity to ensure a biologically active protein is produced. In-house bioactivity testing includes cell proliferation, cytotaxis, calcium flux, secondary cytokine up-regulation, induction of surface antigen expression, and protease assays.

Time (sec)0 7,0006,0005,0004,0003,0002,0001,000

RFU

80,000

40,000

120,000

200,000

160,000

240,000

280,000

0

Substrate only Competitor’s human KLK4GIBCO® Human KLK4

[FGF] (ng/mL)0.001 100101.00.10.01

Mea

n O

D

1.2

1.0

1.4

1.8

1.6

2.0

2.2

0.8

ED50 (ng/mL)

GIBCO = 0.26

Competitor = 0.58

[SCF] (pg/mL)0 105104103102101 106

Mea

n O

D

0.5

0.6

0.7

0.8

0.9

1.0

1.1

1.2

0.4

ED50 (pg/mL)

GIBCO = 548.4

Competitor = 6593.6

1 2 3 4 5

Time (sec)290 380360340320300 400

Fluo

resc

ent

inte

nsity

1.6

1.8

2.0

2.2

2.4

1.4

RANTES SCF

Figure 1. High purity. SDS-PAGE analysis of purified recombinant human RANTES (Cat. No. PHC1054) and mouse SCF (Cat. No. PMC2114).

Figure 4. Calcium flux confirms expected biological response. Calcium flux activation was observed by stimulating Fura-2–loaded rat PMNs with the gibco® chemokine CINC-2β (Cat. No. PRC1564).

Figure 2. More bioactive protein due to exceptional refolding techniques. Proliferation of MO7e cells in response to SCF from gibco® and the competitor (Cat. No. PHC2115). As illustrated by the lower ED50, less gibco® SCF is required to yield a response.

Figure 5. High bioactivity of FGF-basic. Proliferation of Balb/3T3 cells in response to FGF (Cat. No. PHG0024) is demonstrated. gibco® FGF-basic is more active than the competitor’s FGF-basic.

Figure 3. Structural homogeneity. The SDS-PAGE gel was run on Human BMP-2 (Cat. No. PHC7145) under reducing (lanes 2 and 3) and non-reducing (lanes 4 and 5) conditions. Under non-reducing conditions, BMP-2 is seen as a dimer, consistent with biologically active BMP-2.

Figure 6. Protease activity confirms protein functionality. Enzymatic activity of Kallikrein 4 (KLK4) was measured by its ability to cleave fluorogenic peptide substrate Boc-Val-Pro-Arg-AMC. The end product is measured using fluorescence excitation and emission wavelengths of 380 nm and 460 nm, respectively.

Characteristic Test performed Criteria for success Figure

Purity SDS-PAGE >95% purity 1

Refolding Bioassay Bioactive protein 2

Structural homogeneity SDS-PAGE Clean band 3

Biological activity Published bioassay ED50 ≤ other available vendors 4, 5, 6

Stability Freeze/thaw Negligible loss

Endotoxin LAL (Limulus amoebocyte lysate) method <0.1 μg/mg


12


Gibco® Recombinant human FGF-basicFGF-basicFGF-basic is a critical component for maintaining embryonic stem cells in culture in an undifferentiated state. gibco® offers recombinant human FGF-basic, a bioactive protein intended for use in cell culture applications. gibco® comes in two forms:

1) FGF-basic (AA 1-155)—a full-length version2) FGF-basic (AA 10-155)—a truncated version

You’ve trusted gibco® to provide high quality cell culture media and reagents. Now supplement your media with gibco® FGF-basic!

Figure 1—Balb/3T3 cells (mouse fibroblast cell line, ATCC: CCL-163) at constant density were co-cultured with serial dilutions of FGF-basic samples in serum-free medium at 37 oC, 5% CO2 between 18 – 24 hours. The optical density (OD) of each concentration of the testing samples, which is directly proportional to the cell number, was read at 490/650 nm at the end point of incubation. The cell proliferation dose curve was plotted with mean OD vs. FGF-b concentrations (ng/ml).

Figure 2—ED50 of Recombinant human FGF-basic (AA 10-155). The optical density (OD) of each concentration of the testing samples, which is directly proportional to the cell number, was read at 490/650 nm at the end point of incubation. The cell proliferation dose curve was plotted with mean OD vs. FGF-b concentrations (ng/ml).

Ordering InformationProduct Name Species Quantity Cat. no.

FGF-basic (AA 1-155) Recombinant Human Human 100 μg PHG0261

FGF-basic (AA 1-155) Recombinant Human Human 1 mg PHG0263




FGF-basic (AA 10-155) Recombinant Human Human 1 mg PHG0023



FGF-basic (AA 10-155) Recombinant Human (Liquid Form) Human 100 μg PHG0021L





Gibco® LIF RecombinantLIF RecombinantWhile known for its pleiotropic actions, Leukemia Inhibitory Factor (LIF) is most notable for its ability to prevent the differentiation of mouse embry-onic stem cells in culture enabling them to maintain their pluripotency. gibco® offers both recombinant mouse LIF and recombinant human LIF, bioactive proteins intended for use in cell culture applications. You’ve trusted gibco® to provide high quality cell culture media and reagents. Now supplement your media with gibco® LIF!

Mouse embryonic stem cells cultured with gibco® recombinant LIF maintain pluripotency Pluripotency of mouse embryonic stem cells cultured with gibco® LIF has been confirmed by alkaline phosphatase expression, by expression of pluripotency markers, and by the ability of the mouse embryonic stem cells to generate the three germ layers.

Alkaline phosphatase is a marker of undifferentiated embryonic stem cells

Figure 1—a) undifferentiated mouse embryonic stem cells and; b) differentiated mouse embryonic stem cells; and c) mouse embryonic stem cells cultured in 15% KnockOut TM Serum Replacement (KSR) and 10 ng/mL of gibco® mouse LIF.

Figure 2—The dose dependent reduction of MTS within stimulated mouse M1 cells to measure the biological activity of a) gibco® recombinant mouse LIF b) gibco® recombinant human LIF compared to the competitor LIF.

Biological activity of gibco® LIF

Concentration (ng/ml)

1e-4 0.001 0.01 0.1 1 10 1001.7

1.9

2.1

2.3

2.5

2.7

2.9Recombinant LIF

4-P Fit: y = (A - D)/( 1 + (x/C)^B ) + D: A B C D R^2Plot#1 (Competitor: Concentration vs MeanValue) 1.78 1.81 0.253 2.8 0.989Plot#2 (GIBCO: Concentration vs MeanValue) 1.78 1.39 0.138 2.82 0.999

__________Weighting: Fixed

Experiment#1

10/13/2010 10:57:21 AM \\Frd01file03...\MsLIF\mESC\ALP test 10MAR10.pdaSoftMax Pro 5.4

1 of 1


LIF Recombinant Human 100 μg PHC9461

LIF Recombinant Human 10 μg PHC9464

LIF Recombinant Mouse 100 μg PMC4051

LIF Recombinant Mouse 10 μg PMC4054


14


Gibco® Recombinant Growth Factors for use in stem cell applicationsWe are expanding our line of gibco® recombinant proteins with three growth factors important to stem cell research—Thrombopoietin (TPO), BMP-4, and Activin-A. These join other gibco® growth factors and cytokines used in stem cell research, like GM-CSF, TGFα, FGF basic, Stem Cell Factor, TGF-β1, Flt-3 Ligand, IL-2, IL-4, IL-6, and IL-7.

Thrombopoietin (TPO)TPO is a humoral growth factor that is necessary for megakaryocyte proliferation and maturation, as well as for thrombopoiesis. This protein is the ligand for MLP/MPL, the product of myeloproliferative leukemia virus oncogene.


Thrombopoietin, Recombinant Human Human 10μg PHC9514

Thrombopoietin, Recombinant Human Human 100μg PHC9511

Thrombopoietin, Recombinant Human Human 1 mg PHC9513

BMP-4BMP4 is a member of the bone morphogenetic protein family which is part of the transforming growth factor-beta superfamily. Bone morphogenetic proteins were originally identified by an ability of demineralized bone extract to induce endochondral osteogenesis in vivo in an extraskeletal site. This particular family member plays an important role in the onset of endochondral bone formation in humans.


BMP-4, Recombinant Human Human 10 μg PHC9534

BMP-4, Recombinant Human Human 100 μg PHC9531

BMP-4, Recombinant Human Human 1 mg PHC9533

Activin AActivin A is a member of the transforming growth factor-beta superfamily. Activin A plays a role in hormone secretion, neuronal survival, and proliferation.


Activin A, Recombinant Human Human 10 μg PHC9564

Activin A, Recombinant Human Human 100 μg PHC9561

Activin A, Recombinant Human Human 1 mg PHC9563

BMP-7BMP-7 plays a role in the differentiation of mesenchymal cells into bone and cartilage. BMP7 may also be involved in bone homeostasis.

Ordering InformationProduct Name Quantity Cat. no.

BMP-7, Recombinant Human 10 μg PHC9544



new





IL-17FIL-17F is unregulated during the inflammation response. IL-17F has been implicated in the allergic response.

Ordering InformationProduct Name Quantity Cat. no.

IL-17F, Recombinant Human 10 μg PHC9554



Human proteinsProduct Quantity Cat. no.

sCD4 new 10 μg PHS0044

CD40 Ligand/TRAP (soluble)

10 μg PHP0024

CD40 Ligand/TRAP (soluble)

50 μg PHP0025

CNTF 20 μg PHC7015

Cripto new 10 μg PHC8014



Cripto new 1 mg PHC8013

CTGF 20 μg PHG0286

CTLA-Fc 100 μg PHR5001

Dkk-1 new 10 μg PHC9214



Dkk-1 new 1 mg PHC9213

EGF new 10 μg PHG0314



EGF new 1 mg PHG0313

EGF (liquid) new 100 μg PHG0311L

EMAP-II 20 μg PHC1585

ENA-78 10 μg PHC1335

ENA-78 25 μg PHC1336

ENA-78 100 μg PHC1331

ENA-78 1 mg PHC1333

Eotaxin 10 μg PHC1434

Eotaxin 100 μg PHC1431

Eotaxin 1 mg PHC1433

Erythropoietin 500 IU PHC2054

Product Quantity Cat. no.

4-1BBL 20 μg PHC1564

6CKine/SLC 10 μg PHC1474

6CKine/SLC 100 μg PHC1471

6CKine/SLC 1 mg PHC1473

Active Caspase-3 100 units PHZ0014

Activin A 5 μg PHG9014

Activin R1b new 10 μg PHC9284



Activin R1b new 1 mg PHC9283

α-Defensin (NP-1) 20 μg PHC1615

α-Synuclein new 200 μg PHB0044

Angiopoietin-1 10 μg PHC1514

Angiopoietin-2 10 μg PHC1524

BAFF 20 μg PHC1674

BCA-1 (CXCL13) new 20 μg PHC0052

β-Defensin (BD-1) 20 μg PHC1624

B-DNF 10 μg PHC7074

β-NGF new 20 μg PHG0126

β-Synuclein new 200 μg PHB0064

BMP-2 new 10 μg PHC7145



BMP-2 new 1 mg PHC7143

BMP-4 5 μg PHC7914

BMP-7 (active) 10 μg PHC7204

BMP-7 (inactive) 10 μg PHC7104

BMPR1A new 10 μg PHC9244

BMPR1A new 100 μg PHC9241

BMPR1A new 1 mg PHC9243

Cardiotrophin-1 10 μg PHC1594


FGF-4 25 μg PHG0154




FGF-8b 10 μg PHG0274



FGF-8b 1 mg PHG0273







FGF-acidic 10 μg PHG0014

FGF-basic 10 μg PHG0024



FGF-basic aa10–155 (liquid) new

100 μg PHG0021L

FGF-basic 1 mg PHG0023

FGF-basic (full length) 10 μg PHG0264



FGF-basic (full length) 1 mg PHG0263

Flt-3 Ligand new 10 μg PHC9414



Flt-3 Ligand new 1 mg PHC9413


16



Frizzled-1 new 10 μg PHC9384



Frizzled-1 new 1 mg PHC9383





G-CSF 25 μg PHC2035

G-CSF 100 μg PHC2031

G-CSF new 1 mg PHC2033

G-DNF 5 μg PHC7044

G-DNF 10 μg PHC7045

G-DNF 100 μg PHC7041

G-DNF new 1 mg PHC7043

GM-CSF 10 μg PHC2015

GM-CSF 100 μg PHC2011

GM-CSF 1 mg PHC2013

GRO-α 10 μg PHC1065



GRO-α 1 mg PHC1063

GRO-β 10 μg PHC1074

GRO-β (CXCL2,AA-39-107) new

10 μg

PHC9224

GRO-β (CXCL2,AA-39-107) new

100 μg PHC9221

HGF 10 μg PHG0254

IFN-α A 5 x 106 PHC4814

IFN-α A 5 μg PHC4014

IFN-α A/D, Bgl II Hybrid 105 units PHC4044

IFN-α B2 105 units PHC4064

IFN-α D Subtype 106 units PHC4054

IFN-β new 10 units PHC4244

IFN-γ 100 μg PHC4031

IFN-γ 1 mg PHC4033

IGF-II 50 μg PHG9074

IL-1α 5 μg PHC0015



IL-1α 1 mg PHC0013

IL-1β 2 μg PHC0814




IL-1β 1 mg PHC0813


IL-2 10 μg PHC0026

IL-2 40 μg PHC0027

IL-2 100 μg PHC0021

IL-2 1 mg PHC0023

IL-3 10 μg PHC0034

IL-3 50 μg PHC0035

IL-3 100 μg PHC0031

IL-3 1 mg PHC0033

IL-4 10 μg PHC0044

IL-4 25 μg PHC0045

IL-4 100 μg PHC0041

IL-4 1 mg PHC0043

IL-5 10 μg PHC0055

IL-6 5 μg PHC0064

IL-6 10 μg PHC0065

IL-6 25 μg PHC0066

IL-6 100 μg PHC0061

IL-6 1 mg PHC0063

IL-7 5 μg PHC0075

IL-7 25 μg PHC0076

IL-7 100 μg PHC0071

IL-7 1 mg PHC0073

IL-8 (endothelial) 25 μg PHC0084

IL-8 (monocyte) 25 μg PHC0884



IL-8 (monocyte) 1 mg PHC0883

IL-10 5 μg PHC0104

IL-10 10 μg PHC0105

IL-10 100 μg PHC0101

IL-10 1 mg PHC0103

IL-11 10 μg PHC0115

IL-12 new 10 μg PHC1124

IL-12 new 1 mg PHC1123

IL-13 5 μg PHC0134

IL-13 25 μg PHC0135

IL-13 100 μg PHC0131

IL-15 10 μg PHC9154

IL-15 100 μg PHC9151

IL-17 10 μg PHC9174

IL-17 25 μg PHC0174

IL-17 100 μg PHC9171

IL-17 1 mg PHC9173

IL-18/IGIF 25 μg PHC0186

IL-19 10 μg PHC0194

IL-19 100 μg PHC0191

IL-20 10 μg PHC0204

IL-20 25 μg PHC0205

IL-20 100 μg PHC0201


IL-21 10 μg PHC0214

IL-21 25 μg PHC0215

IL-21 100 μg PHC0211

IL-21 1 mg PHC0213








IL-33 new 1 mg PHC9253

IP-10 10 μg PHC1084

IP-10 25 μg PHC1085

IP-10 100 μg PHC1081

IP-10 1 mg PHC1083

ITAC 10 μg PHC1694

ITAC 100 μg PHC1691

Kallikrein 4 new 10 μg PHC9344



Kallikrein 4 new 1 mg PHC9343









KGF-2 25 μg PHC1536

Leptin 1 mg PHP0013

LIF new 10 μg PHC9464

LIF new 100 μg PHC9461

MCP-1 10 μg PHC1014



MCP-2 5 μg PHC1114

MCP-2 new 10 μg PHC1116





M-CSF 10 μg PHC9504

M-CSF 100 μg PHC9501

MDC/STCP-1 20 μg PHC1204

Midkine 20 μg PHC7084

Human proteins, continued

units






MIG 10 μg PHC1604

MIG 1 mg PHC1603

MIG 100 μg PHC1601

MIP-1α 10 μg PHC1104


MIP-1β new 10 μg PHC1034Z

MIP-1β (liquid) 1 mg PHC1033L

MIP-1β (liquid) 100 μg PHC1031L


MIP-3β 20 μg PHC1244

MIP-4 10 μg PHC1254

MIP-5 25 μg PHC1544

Natural Fibronectin new 1 mg PHE0023

Natural Vitronectin new 100 μg PHE0011

Neurotactin/Fractalkine 20 μg PHC1174

Neurotrophic Factor-4 (NT-4)

5 μg PHC7024

Neurturin 10 μg PHC7064

Neutrophil Activation Protein-2 (NAP-2)

2 μg PHC1094

Neutrophil Activation Protein-2 (NAP-2)

10 μg PHC1095

Noggin new 20 μg PHC1506

Oncostatin M 10 μg PHC5015

Osteoprotegerin (OPG) 50 μg PHC1684

PDGF-AA 10 μg PHG0035

PDGF-AB 10 μg PHG0134


PDGF-BB 5 μg PHG0044




PDGF-BB 1 mg PHG0043

PF4 new 20 μg PHC7056

PIGF-1 20 μg PHG0296

Pleiotrophin (PTN, HARP) 20 μg PHC7075

RANK Ligand 10 μg PHP0034

RANTES 10 μg PHC1054



RANTES 1 mg PHC1053

SCF/c-Kit Ligand 10 μg PHC2115



SCF/c-Kit Ligand 1 mg PHC2113

SDF-1α new 10 μg PHC1344



SDF-1α new 1 mg PHC1343

SDF-1β 10 μg PHC1364

TARC 20 μg PHC1264

TECK 20 μg PHC1625

TGF-α 100 μg PHG0051


TGF-β1 1 mg PHG9203

TGF-β1 5 μg PHG9204

TGF-β1 new 10 μg PHG9214

TGF-β1 new 100 μg PHG9211





TIMP-1 new 10 μg PHC8024

TIMP-1 new 100 μg PHC8021

TIMP-1 new 1 mg PHC8023

TNF-α 10 μg PHC3015

TNF-α (liquid) 10 μg PHC3015L



TNF-α 1 mg PHC3013

TNF-RI 20 μg PHR3015

TNF-RII 20 μg PHR3025

TRAIL 50 μg PHC1634

VEGF new 10 μg PHC9394

VEGF new 100 μg PHC9391

VEGF new 1 mg PHC9393

Wnt-1 new 10 μg PHC1804


18



Ciliary Neurotrophic Factor (CNTF) 25 μg PRC7015

GM-CSF 10 μg PRC2015



GRO/KC/CINC-1 25 μg PRC1066

GRO-β/MIP-2 100 μg PRC1071

GRO-β/MIP-2 10 μg PRC1074

IFN-α 2 x 105 units PRC4014

IFN-β 3 x 105 units PRC4024

IFN-γ 10 μg PRC4034

IFN-γ 100 μg PRC4035

IFN-γ 1 mg PRC4033

IL-1α 10 μg PRC0014

IL-1α 100 μg PRC0011

IL-1β 10 μg PRC0814



IL-1β 1 mg PRC0813

IL-2 10 μg PRC0024

IL-2 100 μg PRC0021

IL-3 10 μg PRC0034

IL-3 100 μg PRC0031


IL-5 10 μg PRC0054

IL-5 100 μg PRC0051

IL-5 1 mg PRC0053

IL-6 10 μg PRC0064

IL-6 100 μg PRC0061

IL-6 1 mg PRC0063

IL-10 10 μg PRC0104

IL-13 10 μg PRC0134

IL-13 100 μg PRC0131

IL-13 1 mg PRC0133

IL-18/IGIF 10 μg PRC0184

IL-18/IGIF 100 μg PRC0181

MCP-1 10 μg PRC1014



RANTES 20 μg PRC1054

TNF-α 10 μg PRC3014

TNF-α 100 μg PRC3015

TNF-α 1 mg PRC3013

VEGF 10 μg PRG0114

VEGF 25 μg PRG0115

VEGF 100 μg PRG0111

VEGF 1 mg PRG0113

Rat proteins






GM-CSF 10 μg PSC2014



IFN-γ 10 μg PSC4034

IFN-γ 100 μg PSC4031

IL-1α 10 μg PSC0014

IL-1α 100 μg PSC0011

IL-2 10 μg PSC0024

IL-2 100 μg PSC0021

IL-4 10 μg PSC0044

IL-4 25 μg PSC0045

IL-4 100 μg PSC0041

IL-6 10 μg PSC0064

IL-6 100 μg PSC0061

IL-8 10 μg PSC0884

IL-8 100 μg PSC0881

IL-10 10 μg PSC0104

IL-10 100 μg PSC0101

IL-15 10 μg PSC0154

IL-15 100 μg PSC0151

TNF-α 10 μg PSC3014

TNF-α 100 μg PSC3011

Swine proteins


20


Have your cytokines, chemokines, and growth factors been referenced in publications?

Proven CompatibilityNg F et al. (2008) PDGF, TGF-β, and FGF signaling is important for differentiation and growth of mesenchymal stem cells (MSCs): transcriptional profiling can identify markers and signaling pathways important in differentiation of MSCs into adipogenic, chondrogenic, and osteogenic lineages. Blood 112:295–307.

Human EGFShimada T et al. (2009) Development of targeted therapy with paclitaxel incorporated into EGF-conjugated nanoparticles. Anticancer Res 29:1009–1014.

Human FGF-basicLevenstein M et al. (2006) Basic fibroblast growth factor support of human embryonic stem cell self-renewal. Stem Cells 24:568–574.

Jerebtsova M et al. (2007) A novel role of fibroblast growth factor-2 and pentosan polysulfate in the pathogenesis of intestinal bleeding in mice. Am J Physiol Heart Circ Physiol 292:743–750.

Lu S et al. (2008) Biologic properties and enucleation of red blood cells from human embryonic stem cells. Blood 112:4475–4484.

Karumbayaram S et al. (2009) Human embryonic stem cell-derived motor neurons expressing SOD1 mutants exhibit typical signs of motor neuron degeneration linked to ALS. Dis Model Mech 2:189–195.

Zhou J et al. (2009) mTOR supports long-term self-renewal and suppresses mesoderm and endoderm activities of human embryonic stem cells. Proc Natl Acad Sci U S A 106:7840–7845.

Human G-CSFMatsui W et al. (2005) Requirement for myeloid growth factors in the differentiation of acute promyelocytic leukaemia. Br J Haematol 128:853–862.

Human IL-2Zeddou M et al. (2005) Prostaglandin E2 induces the expression of func-tional inhibitory CD94/NKG2A receptors in human CD8+ T lymphocytes by a cAMP-dependent protein kinase A type I pathway. Biochem Pharmacol 70:714–724.

Kemper C et al. (2003) Activation of human CD4+ cells with CD3 and CD46 induces a T regulatory cell 1 phenotype. Nature 421:388–392.

Human IL-4Pietrella D et al. (2008) A Candida albicans mannoprotein deprived of its mannan moiety is efficiently taken up and processed by human dendritic cells and induces T cell activation without stimulating proinflammatory cytokine production. Infect Immun 76:4359–4367.

Human IL-21de Totero B et al. (2008) The opposite effects of IL-15 and IL-21 on CLL B cells correlate with differential activation of the JAK/STAT and ERK1/2 pathways. Blood 111:517–524.

Human MIP-1αBluman EM et al. (1996) Human natural killer cells produce abundant macrophage inflammatory protein-1 alpha in response to monocyte-derived cytokines. J Clin Invest 97:2722–2727.

Human PDGF-BBHou S et al. (2002) In adenovirus type 12 tumorigenic cells, major histocompatibility complex class I transcription shutoff is overcome by induction of NF-κB and relief of COUP-TFII repression. J Virol 76:3212–3220.

Human SCF/c-kit ligandDenis MM et al. (2005) Escaping the nuclear confines: signal-dependent pre-mRNA splicing in anucleate platelets. Cell 122:379–391.

Lindberg SR et al. (2005) The leukemia-associated ETO homologues are differently expressed during hematopoietic differentiation. Exp Hematol 33:189–198.

Mouse GM-CSFJamur MC et al. (2005) Identification and characterization of undifferentiated mast cells in mouse bone marrow. Blood 105:4282–4289.

Mouse IL-6Selander C et al. (2009) TLR2/MyD88-dependent and -independent activation of mast cell IgE responses by the skin commensal yeast Malassezia sympodialis. J Immunol 182:4208–4216.

Mouse IL-7Alter-Wolf S et al. (2009) Deviation of the B cell pathway in senescent mice is associated with reduced surrogate light chain expression and altered immature B cell generation, phenotype, and light chain expression. J Immunol 182:138–147.

Mouse PDGF-BBClair T et al. (2003) Autotaxin hydrolyzes sphingosylphosphorylcholine to produce the regulator of migration, sphingosine-1-phosphate. Cancer Res 63:5446–5453.

Mouse SCFAlter-Wolf S et al. (2009) Deviation of the B cell pathway in senescent mice is associated with reduced surrogate light chain expression and altered immature B cell generation, phenotype, and light chain expression. J Immunol 182:138–147.





Notes:


22


Notes:


Chapter 3 Basal Media

continued

Reduced-Serum Media at a Glance . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25

GlutaMAX™ Media at a Glance . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 26

HEPES Media at a Glance . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 27

Phenol Red–free Media at a Glance. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 28

Basal Medium Eagle . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 28

BGJb Medium (Fitton-Jackson Modification) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 28

Brinster’s BMOC-3 Medium . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 29

Cryopreservation Medium . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 29

CMRL Medium. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 29

CO2-Independent Medium . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 29

Dulbecco’s Modified Eagle Media (DMEM) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 30

Advanced DMEM . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 30

DMEM (high glucose) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 30

DMEM (low glucose). . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 32

DMEM (no glucose) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 32

DMEM, powder (low glucose) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 32

DMEM Nutrient Mixture F-12 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 33

F-10 Nutrient Mixtures. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 34

F-12 Nutrient Mixtures. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 34

Glasgow Minimum Essential Medium . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35

Grace’s Insect Cell Culture Media . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35

Improved MEM Zn++ . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 36

IPL-41 Insect Medium . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 36

Iscove’s Modified Dulbecco’s Media . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 36

Leibovitz’s L-15 Media . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 37

McCoy’s 5A Medium (Modified) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 37

MCDB 131 Medium . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 37

Media 199 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 38

Minimum Essential Media (MEM) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 39

Advanced MEM (Minimum Essential Medium) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 40

Minimum Essential Medium (MEM) with Earle’s Salts . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 40

Minimum Essential Medium (MEM) with Hanks’ Salts . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 42


24


Modified Eagle Media . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 42

Medium NCTC-109 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 42

F-10 Nutrient Mixtures . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 42

F-12 Nutrient Mixtures . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 42

Opti-MEM® I Reduced-Serum Media . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 43

Lipofectamine™ 2000 Transfection Reagent . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 43

RPMI Media 1640 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 44

Schneider’s Drosophila Medium . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 46

Waymouth’s MB 752/1 Medium . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 46

Williams’ Medium E . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 46

Media Preparation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 47

E-Z Hold™ Plastic Bottles . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 49

Stak Pack™ Accessories . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 49

Media Formulations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 50

gibco® cell culture catalogueBasal M

edia3



Reduced-serum Media at a GlanceReduced-serum media significantly reduce serum requirements, which helps decrease variability and extend the life of a serum lot. Advanced media formulations reduce FBS supplementation by 50–90%, and Opti-MEM® Media reduce FBS supplementation by at least 50%, with no change in cell growth, promotion, morphology, or function.

For your convenience, this page consolidates all gibco® reduced-serum media in a concise, easy-to-use table. You can quickly scan it to determine what you need and where the product is fully described in this catalogue. For performance testing data of Advanced media go to www.invitrogen.com/advanced.

Reduced-serum MediaProduct Quantity Cat. no. Page

Advanced DMEM (Dulbecco’s Modified Eagle Medium) (1X), liquid

Contains 4,500 mg/L D-glucose, non-essential amino acids (NEAA), and 110 mg/L of sodium pyruvate, but no L-glutamine

500 ml 10 × 500 ml (case)

12491-015 12491-023 68

Advanced DMEM/F-12 (1X), liquid, 1:1 Contains 3,151 mg/L D-glucose, non-essential amino acids (NEAA), and 110 mg/L sodium pyruvate, but no L-glutamine

500 ml 10 × 500 ml (case)

12634-010 12634-028 71

Advanced MEM (Minimum Essential Medium) (1X), liquid

Contains 110 mg/L of sodium pyruvate and non-essential amino acids (NEAA), but no L-glutamine

500 ml 10 × 500 ml (case)

12492-013 12492-021 78

Advanced RPMI Medium 1640 (1X), liquid Contains 2,000 mg/L D-glucose, non-essential amino acids (NEAA), and 110 mg/L sodium pyruvate, but no L-glutamine

500 ml 10 × 500 ml (case)

12633-012 12633-020 83

Opti-MEM® I Reduced-Serum Medium (1X), liquid

Contains HEPES buffer, 2,400 mg/L sodium bicarbonate, hypoxanthine, thymidine, sodium pyruvate, L-glutamine, trace elements, growth factors, and phenol red reduced to 1.1 mg/L

100 ml 10 x 500 ml (case)

31985-047 31985-054 81

Opti-MEM® I Reduced-Serum Medium (1X), liquid (w/o phenol red)

Contains HEPES buffer, 2,400 mg/L sodium bicarbonate, hypoxanthine, thymidine, sodium pyruvate, L-glutamine, trace elements, and growth factors, but no phenol red

500 ml 11058-021 81

Opti-MEM® I Reduced-Serum Medium (1X), liquid (with GlutaMAX™-I)

Contains GlutaMAX™-I substituted on a molar equivalent basis for L-glutamine, HEPES buffer, 2,400 mg/L sodium bicarbonate, hypoxanthine, thymidine, sodium pyruvate, L-glutamine, trace elements, growth factors, and phenol red reduced to 1.1 mg/L

500 ml 10 × 500 ml (case)

51985-026 51985-042 81

Opti-MEM® I Reduced-Serum Medium, powder A modification of MEM (Eagle’s) Contains HEPES buffer, hypoxanthine, thymidine, sodium pyruvate, L-glutamine, trace elements, growth factors, and phenol red reduced to 1.1 mg/L, but no sodium bicarbonate

10 × 1 L

22600-134 81

For more information on classifications, production, or preparation of the products in this chapter, please see pages 47 and 48.


26


GlutaMAX™ Media at a Glance

GlutaMAX™ MediaProduct Quantity Cat. no. Page

Dulbecco’s Modified Eagle Medium (DMEM) (1X), liquid Low glucose Contains sodium pyruvate

500 ml 10 × 500 ml (case)

21885-025 21885-108 70

Dulbecco’s Modified Eagle Medium (DMEM) (1X), liquid High glucose Contains sodium pyruvate

500 ml 10 × 500 ml (case)

31966-021 31966-047 68

Dulbecco’s Modified Eagle Medium (DMEM) (1X), liquid High glucose Contains no sodium pyruvate

500 ml 10 × 500 ml (case)

61965-026 61965-059 68

Dulbecco’s Modified Eagle Medium (DMEM) (1X), liquid High glucose Contains HEPES buffer but no sodium pyruvate

500 ml 10 × 500 ml (case)

32430-027 32430-100 68

DMEM/F-12, (1X) liquid, 1:1 500 ml 10 × 500 ml (case)

31331-028 31331-093 68

F-12 Nutrient Mixture (Ham) (1X), liquid 500 ml 10 × 500 ml (case)

31765-027 31765-068 72

Iscove’s Modified Dulbecco’s Medium (IMDM) (1X), liquid 500 ml 10 × 500 ml (case)

31980-022 31980-048 74

Minimum Essential Medium (MEM) alpha (1X), liquid Contains no ribonucleosides or deoxyribonucleosides 500 ml 10 × 500 ml (case)

32561-029 32561-094 79

Minimum Essential Medium (MEM) alpha (1X), liquid Contains ribonucleosides and deoxyribonucleosides 500 ml 10 × 500 ml (case)

32571-028 32571-093 79

Minimum Essential Medium (MEM), liquid Contains Earle’s salts 500 ml 10 × 500 ml (case)

41090-028 41090-093 79

Minimum Essential Medium (MEM), liquid Contains Earle’s salts and HEPES buffer 500 ml 10 × 500 ml (case)

42360-024 42360-081 74

Opti-MEM® I Reduced-Serum Medium (1X), liquid 500 ml 10 × 500 ml (case)

51985-026 51985-042 81

RPMI Medium 1640 (1X), liquid 500 ml 10 × 500 ml (case)

61870-010 61870-044 83

RPMI Medium 1640 (1X), liquid Contains HEPES buffer 500 ml 10 × 500 ml (case)

72400-021 72400-054 83

For stability data of GlutaMAX™ -I vs. L-glutamine, see Appendix I, page 202.

GlutaMAX™ Media have increased stability, minimize toxic ammonia build-up, and improve cell viability. They are standard cell culture formulations that contain L-glutamine as a stabilized dipeptide.

For your convenience, this page consolidates all gibco® GlutaMAX™ Media in a concise, easy-to-use table. You can quickly scan it to determine what you need and where the product is fully described in this catalogue.


edia3



HEPES Media at a Glance

HEPES MediaProduct Quantity Cat. no. Page

Dulbecco’s Modified Eagle Medium (DMEM) (1X), liquid (high glucose)

Contains 4,500 mg/L D-glucose, L-glutamine, and 25 mM HEPES buffer, but no sodium pyruvate

500 ml 10 × 500 ml (case)

42430-025 42430-082 68


Contains 4,500 mg/L D-glucose, L-glutamine, and 25 mM HEPES buffer, but no sodium pyruvate or phenol red 500 ml 21063-029 69

Dulbecco’s Modified Eagle Medium (DMEM) (1X), liquid (low glucose)

Contains 1,000 mg/L D-glucose, L-glutamine, 25 mM HEPES buffer, and 110 mg/L sodium pyruvate

500 ml 10 × 500 ml (case)

22320-022 22320-030 69


Contains GlutaMAX™-I substituted on a molar equivalent basis for L-glutamine and 25 mM HEPES buffer, but no sodium pyruvate

500 ml 10 × 500 ml (case)

32430-027 32430-100 70

DMEM/F-12 (1X), liquid, 1:1 Contains 15 mM HEPES buffer and L-glutamine 500 ml 10 × 500 ml (case)

31330-038 31330-095 71

DMEM/F-12 (1X), liquid, 1:1 Contains 15 mM HEPES buffer and L-glutamine, but no phenol red 500 ml 10 × 500 ml (case)

11039-021 11039-047 71

DMEM/F-12, powder, 1:1 Contains 15 mM HEPES buffer and L-glutamine, but no sodium bicarbonate 1 L 1 × 10 L

42400-010 42400-028 71

F-10 Nutrient Mixture (Ham) (1X), liquid Contains 25 mM HEPES buffer and L-glutamine 500 ml 22390-025 72

Iscove’s Modified Dulbecco’s Medium (IMDM) (1X), liquid

Contains L-glutamine, 25 mM HEPES buffer, and 3,024 mg/L sodium bicarbonate, but no α-thioglycerol or 2-mercaptoethanol

500 ml 10 × 500 ml (case)

21980-032 21980-065 74


Contains GlutaMAX™-I substituted on a molar equivalent basis for L-glutamine, 25 mM HEPES buffer, and 3,024 mg/L sodium bicarbonate

500 ml 10 × 500 ml (case)

31980-022 31980-048 74


Contains L-glutamine, 25 mM HEPES buffer, and 3,024 mg/L sodium bicarbonate, but no α-thioglycerol, 2-mercaptoethanol, or phenol red 500 ml 21056-023 74

Iscove’s Modified Dulbecco’s Medium (IMDM), powder

Contains L-glutamine and 25 mM HEPES buffer, but no sodium bicarbonate, α-thioglycerol, or 2-mercaptoethanol

10 × 1 L 1 × 10 L

42200-014 42200-030 74

McCoy’s 5A Medium (modified) (1X), liquid Contains L-glutamine and 25 mM HEPES buffer 500 ml 10 × 500 ml (case)

22330-021 22330-070 75

Medium 199 (1X), liquid Contains Earle’s salts, 2,200 mg/L sodium bicarbonate, L-glutamine, and 25 mM HEPES buffer

500 ml 10 × 500 ml (case)

22340-020 22340-087 76

Medium 199 (1X), liquid Contains Hanks’ salts, 25 mM HEPES buffer, and L-glutamine 500 ml 10 × 500 ml (case)

22350-029 22350-078 76

Minimum Essential Medium (MEM) (1X), liquid

Contains Earle’s salts and 25 mM HEPES buffer, but no L-glutamine 500 ml 10 × 500 ml (case)

32360-026 32360-034 79

Minimum Essential Medium (1X MEM), liquid Contains GlutaMAX™-I substituted on a molar equivalent basis for L-glutamine, Earle’s salts, and 25 mM HEPES buffer

500 ml 10 × 500 ml (case)

42360-024 42360-081 79

RPMI Medium 1640 (1X), liquid Contains 25 mM HEPES buffer and L-glutamine 100 ml 500 ml

10 × 500 ml (case) 20 × 100 ml (case)

52400-017 52400-025 52400-041 52400-058

83

RPMI Medium 1640 (1X), liquid Contains GlutaMAX™-I substituted on a molar equivalent basis for L-glutamine and 25 mM HEPES buffer

100 ml 500 ml

10 × 500 ml (case) 20 × 100 ml (case)

72400-013 72400-021 72400-054 72400-112

83

RPMI Medium 1640, powder Contains L-glutamine and 25 mM HEPES buffer, but no sodium bicarbonate 1 L 1 × 10 L

13018-015 13018-031 83

HEPES-buffered media resist fluctuations in pH caused by changes in cellular metabolism and culture environment. They are recommended for stabilizing the pH of sensitive cell culture systems.

For your convenience, this page consolidates all gibco® HEPES media in a concise, easy-to-use table. You can quickly scan it to determine what you need and where the product is fully described in this catalogue.


28


Basal Medium Eagle (BME)Basal Medium Eagle is well suited for use with diploid or primary mammalian cell cultures.For product formulation please refer to the tables at the end of this chapter.

Basal Medium Eagle (BME) (1X), liquidRecommended storage conditions: 2°C to 8°C.Intended use: In vitro diagnostic (IVD).

Ordering InformationQuantity Cat. no.

500 ml 41010-026

Contains Earle’s salts, but no L-glutamine.

Phenol Red–free Media at a Glance

Phenol Red–free MediaProduct Quantity Cat. no. Page

DMEM (1X), liquid (low glucose) Contains 1.0 g/L D-glucose and 110 mg/L sodium pyruvate (without L-glutamine)

500 ml 10 × 500 ml (case)

11880-028 11880-036

70

DMEM (1X), liquid (high glucose) Contains 4.5 g/L D-glucose, L-glutamine, and 25 mM HEPES buffer 500 ml 21063-029 68

DMEM (1X), liquid (high glucose) Contains 4.5 g/L D-glucose (without L-glutamine) 500 ml 10 × 500 ml (case)

31053-028 11960-085

68

DMEM/F-12 (1X), liquid, 1:1 Contains L-glutamine and 15 mM HEPES buffer 500 ml 10 × 500 ml (case)

11039-021 11039-047

71

DMEM/F-12 (1X), liquid, 1:1 Contains L-glutamine 500 ml 21041-025 71

Iscove’s Modified Dulbecco’s Media (IMDM) (1X), liquid Contains L-glutamine 500 ml 21056-023 74

Improved MEM Zn++ Option M (1X) (Richter’s Modification), liquid

500 ml 10373-017 74

Leibovitz’s L-15 Medium (1X), liquid Contains L-glutamine 500 ml 21083-027 75

Medium 199 (1X), liquid Contains Earle’s salts, L-glutamine, and sodium bicarbonate 500 ml 11043-023 76

MEM α Medium (1X), liquid Contains L-glutamine, ribonucleosides, and deoxyribonucleosides 500 ml 41061-029 78

MEM (1X), liquid Contains Earle’s salts, but no L-glutamine 500 ml 51200-038 79

Modified Eagle Medium (MEM) (2X), liquid 500 ml 21935-028 80

Opti-MEM® I Reduced-Serum Medium (1X), liquid 500 ml 11058-021 81

RPMI Medium 1640 (1X), liquid Contains L-glutamine 500 ml 11835-063 83

Ideal for cell labeling experiments, phenol red–free media allow specific applications not possible with classical media. Phenol red, which is used as a pH indicator, can exhibit estrogen-like properties in some cells, and may be toxic to certain cell types. Gibco® phenol red–free media are nontoxic and exhibit no estrogen-like activity in cell experimentation.

For your convenience, this page consolidates all gibco® phenol red–free media in a concise, easy-to-use table. You can quickly scan it to deter-mine what you need and where the product is fully described in this catalogue.

BGJb Medium (Fitton-Jackson Modification)BGJb Medium was formulated for the growth of fetal rat long bones in a chemically defined medium.For product formulation please refer to the tables at the end of this chapter.

BGJb Medium (Fitton-Jackson Modification) (1X), liquidRecommended storage conditions: 2°C to 8°C.Intended use: In vitro diagnostic (IVD).


500 ml 12591-038

Contains L-glutamine.


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Brinster’s BMOC-3 Medium

Brinster’s BMOC-3 Medium, liquidFor product formulation please refer to the tables at the end of this chapter.Recommended storage conditions: 2°C to 8°C.


100 ml 11126-034

Contains bovine serum albumin, but no antibiotics.

Cryopreservation MediumRecovery™ Freezing Medium is suitable for the consistent cryo preservation of a broad spectrum of mammalian cells.

Recovery™ Freezing MediumRecommended storage conditions: –5°C to –20°C.


50 ml 12648-010

Contains DMEM, fetal bovine serum, calf serum, and 10% dimethylsulfoxide (DMSO).

CMRL MediumCMRL Medium is well suited for growth of Earle’s “L” cells. It is especially useful for cloning monkey kidney cell cultures and for growth of other mammalian cell lines when enriched with horse or calf serum.For product formulation please refer to the tables at the end of this chapter.

CMRL Medium, 1066 (1X), liquidRecommended storage conditions: 2°C to 8°C.Intended use: In vitro diagnostic (IVD).


500 ml 21530-027

Contains no L-glutamine.

CMRL Medium, 1066 (10X), liquidRecommended storage conditions: 2°C to 8°C.Intended use: In vitro diagnostic (IVD).


500 ml 21540-026


CO2-Independent MediumThis medium contains a buffering system that maintains a stable pH for extended periods of time under atmospheric CO2.

CO2-Independent Medium (1X), liquidA non-HEPES proprietary medium suitable for supporting cell growth for a variety of epithelial, fibroblast, and lymphoid cell lines in atmospheric conditions.1 It is ideal for transporting cells or tissue and for the handling of mouse embryos in atmospheric conditions. Not intended for CO2-based mouse embryo culture.Recommended storage conditions: 2°C to 8°C, in the dark.

Reference1. Battista, P.J. and Weiss, S.A. (1991) FOCUS® 13, 110.


500 ml 18045-054



30


Dulbecco’s Modified Eagle Media (DMEM)Dulbecco’s Modified Eagle Media are well suited for supporting the growth of a broad spectrum of mammalian cell lines. Use the comparison tables as quick references for identifying the catalogue number of the product that’s best for your application.

Advanced DMEM (1X), liquidRecommended storage conditions: 2°C to 8°C, in the dark.

ReferencePrice, P. and Staines, D. (2004) Advanced Media: Reduced Sera Solutions, Quest 3, 25–30.


500 ml 12491-015

10 × 500 ml (case) 12491-023

Contains 4,500 mg/L D-glucose, non-essential amino acids (NEAA), and 110 mg/L of sodium pyruvate, but no L-glutamine.

DMEM (1X), liquid (high glucose)For product formulation please refer to the tables at the end of this chapter.Recommended storage conditions: 2°C to 8°C.Intended use: In vitro diagnostic (IVD).


500 ml 41965-039

10 × 500 ml (case) 41965-062

1,000 ml 41965-047

6 × 1,000 ml (case) 41965-120

Contains 4,500 mg/L D-glucose and L-glutamine, but no sodium pyruvate.

Quantity Cat. no.

500 ml 41966-029

10 × 500 ml (case) 41966-052

Contains 4,500 mg/L D-glucose, L-glutamine, and 110 mg/L sodium pyruvate.

Quantity Cat. no.

500 ml 42430-025

10 × 500 ml (case) 42430-082

Contains 4,500 mg/L D-glucose, L-glutamine, and 25 mM HEPES buffer, but no sodium pyruvate.

Quantity Cat. no.

500 ml 21063-029

Contains 4,500 mg/L D-glucose, L-glutamine, and 25 mM HEPES buffer, but no sodium pyruvate or phenol red.

Quantity Cat. no.

500 ml 11971-025

Contains 4,500 mg/L D-glucose and L-glutamine, but no sodium phosphate or sodium pyruvate.

Quantity Cat. no.

1 × 10 L 52100-039

Contains 4,500 mg/L D-glucose and L-glutamine, but no sodium pyruvate or sodium bicarbonate.

Quantity Cat. no.

1 × 10 L 12800-116

Contains 4,500 mg/L D-glucose, L-glutamine, and 110 mg/L sodium pyruvate, but no sodium bicarbonate.

Quantity Cat. no.

500 ml 11960-044

10 × 500 ml (case) 11960-085

Contains 4,500 mg/L D-glucose, but no L-glutamine or sodium pyruvate.

Quantity Cat. no.

500 ml 21969-035

Contains 4,500 mg/L D-glucose and 110 mg/L sodium pyruvate, but no L-glutamine.

Quantity Cat. no.

500 ml 31053-028

Contains 4,500 mg/L D-glucose, but no L-glutamine, sodium pyruvate, or phenol red.

Quantity Cat. no.

500 ml 21013-024

Contains 4,500 mg/L D-glucose, but no L-glutamine, sodium pyruvate, L-methionine, or L-cystine.


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Quantity Cat. no

500 ml 21068-028

Contains 4,500 mg/L D-glucose, but no L-glutamine, sodium pyruvate, or calcium chloride.

Quantity Cat. no.

500 ml 31966-021

10 × 500 ml (case) 31966-047

A GlutaMAX™-I medium. Contains GlutaMAX™-I substituted on a molar equivalent basis for L-glutamine, 4,500 mg/L D-glucose, and 110 mg/L sodium pyruvate.

Quantity Cat. no.

500 ml 32430-027

10 × 500 ml (case) 32430-100

Contains GlutaMAX™-I substituted on a molar equivalent basis for L-glutamine, 4,500 mg/L D-glucose, and 25 mM HEPES buffer, but no sodium pyruvate.

Quantity Cat. no.

500 ml 61965-026

10 × 500 ml (case) 61965-059

Contains GlutaMAX™-I substituted on a molar equivalent basis for L-glutamine and 4,500 mg/L D-glucose, but no sodium pyruvate.

Comparison of Dulbecco’s Modified Eagle Media (DMEM), High Glucose (4,500 mg/L)Base cat. no. 41965* 41966 42430 21063 11971 52100* 12800

With L-glutamine

With Na+ pyruvate (110 mg/L) ● ●

With HEPES buffer (25 mM) ● ●

Without phenol red ●

Without Na+ phosphate ●

Powder † ● ●

Without L-glutamine 11960* 21969 31053 32430 61965 31966 21068 12491

With GlutaMAX™-I ‡ ● ● ● ●

With HEPES buffer (25 mM) ●

With Na+ pyruvate (110 mg/L) ● ●


Without CaCl2 ●

Without cystine ●

Without methionine ●

* Standard formulation, as published in Dulbecco, R., Freeman, G. (1959) Virology 8, 396. Smith, J.D., Freeman, G., Vogt, M., and Dulbecco, R. (1960) Virology 12, 185. Tissue Culture Standards Committee, In Vitro 6:2, 93. † Powdered formulations do not contain sodium bicarbonate. ‡ GlutaMAX™-I supplement is L-alanyl-L-glutamine, a glutamine dipeptide, which can be used in place of L-glutamine in culture media. It is substituted on an equimolar basis for L-glutamine.

Use this comparison table as a quick reference for identifying the base catalogue number of the product that’s best for your application.

For the exact composition of each product, please refer to the formulation tables starting on page 88.


32


DMEM (1X), liquid (low glucose)For product formulation please refer to the tables at the end of this chapter.Recommended storage conditions: 2°C to 8°C.Intended use: In vitro diagnostic (IVD).


500 ml 31885-023

10 × 500 ml (case) 31885-049

Contains 1,000 mg/L D-glucose, L-glutamine, and 110 mg/L sodium pyruvate.

Quantity Cat. no.

500 ml 22320-022

Contains 1,000 mg/L D-glucose, L-glutamine, 25 mM HEPES buffer, and 110 mg/L sodium pyruvate.

Quantity Cat. no.

500 ml 11880-028

Contains 1,000 mg/L D-glucose and 110 mg/L sodium pyruvate, but no L-glutamine or phenol red.

Intended use: In vitro diagnostic (IVD).Quantity Cat. no.

500 ml 21885-025

10 × 500 ml (case) 21885-108

A GlutaMAX™-I medium. Contains GlutaMAX™-I substituted on a molar equivalent basis for L-glutamine, 1,000 mg/L D-glucose, and 110 mg/L sodium pyruvate.

DMEM (1X), liquid (no glucose)Recommended storage conditions: 2°C to 8°C.


500 ml 11966-025

Contains L-glutamine, but no glucose or sodium pyruvate.

DMEM, powder (low glucose)Recommended storage conditions: 2°C to 8°C.Intended use: In vitro diagnostic (IVD).


1 × 10 L 31600-083

1 × 50 L 31600-091

Contains 1,000 mg/L D-glucose, L-glutamine, and 110 mg/L sodium pyruvate, but no sodium bicarbonate.

Comparison of Dulbecco’s Modified Eagle Media (DMEM), low glucose (1,000 mg/L)

Base cat. no. 31885* 11966 22320 31600*

With L-glutamine


Without glucose ●

Powder † ●

Without L-glutamine 11880 21885

With GlutaMAX™-I § ●


* Standard formulation, as published in Dulbecco, R., Freeman, G. (1959) Virology 8, 396. Smith, J.D., Freeman, G., Vogt, M., and Dulbecco, R. (1960) Virology 12, 185. Tissue Culture Standards Committee, In Vitro 6:2, 93. † Powdered formulations do not contain sodium bicarbonate. § GlutaMAX™-I supplement is L-alanyl-L-glutamine, a glutamine dipeptide, which can be used in the place of L-glutamine in culture media. It is substituted on an equimolar basis for L-glutamine.




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DMEM Nutrient Mixture F-12 (DMEM/F-12) 1:1

Advanced DMEM/F-12 (1X), liquid, 1:1 Recommended storage conditions: 2° to 8°C, in the dark.

ReferencePrice, P. and Staines, D. (2004) Advanced Media: Reduced Sera Solutions, Quest 3, 25–30.www.invitrogen.com/advanced


500 ml 12634-010

10 × 500 ml (case) 12634-028

Contains 3,151 mg/L D-glucose, non-essential amino acids (NEAA), and 110 mg/L sodium pyruvate, but no L-glutamine.

DMEM/F-12 (1X), liquid, 1:1Recommended storage conditions: 2°C to 8°C.Intended use: In vitro diagnostic (IVD).For product formulation please refer to the tables at the end of this chapter.


500 ml 31330-038

10 × 500 ml (case) 31330-095

10 L 11330-107

Contains 15 mM HEPES buffer and L-glutamine.

Quantity Cat. no.

500 ml 11039-021

10 × 500 ml (case) 11039-047

Contains 15 mM HEPES buffer and L-glutamine, but no phenol red.

Quantity Cat. no.

500 ml 21041-025

Contains L-glutamine, but no HEPES buffer or phenol red.

Quantity Cat. no.

500 ml 31331-028

10 × 500 ml (case) 31331-093

A GlutaMAX™-I medium. Contains GlutaMAX™-I substituted on a molar equivalent basis for L-glutamine, but no HEPES buffer.


34


F-10 Nutrient MixturesF-10 Nutrient Mixture (Ham) was formulated for serum-free growth of Chinese hamster ovary (CHO) cells and growth of various mammalian cell lines with serum supplementation.For product formulation please refer to the tables at the end of this chapter.

F-10 Nutrient Mixture (Ham) (1X), liquidRecommended storage conditions: 2°C to 8°C.Intended use: In vitro diagnostic (IVD).


500 ml 31550-023


Quantity Cat. no.

500 ml 22390-025


F-12 Nutrient MixturesF-12 Nutrient Mixture was formulated for single-cell plating of near-diploid Chinese hamster ovary (CHO) cells.

F-12 Nutrient Mixture (Ham) (1X), liquidRecommended storage conditions: 2°C to 8°C.Intended use: In vitro diagnostic (IVD).For product formulation please refer to the tables at the end of this chapter.


500 ml 21765-029

10 × 500 ml (case) 21765-037

1,000 ml 11765-047


Quantity Cat. no.

500 ml 31765-027

10 × 500 ml (case) 31765-068

A GlutaMAX™-I medium. Contains GlutaMAX™-I substituted on a molar equivalent basis for L-glutamine.

F-12 Nutrient Mixture (Ham), powderRecommended storage conditions: 2°C to 8°C.Intended use: In vitro diagnostic (IVD).For product formulation please refer to the tables at the end of this chapter.


10 × 1 L 21700-026

1 × 50 L 21700-018

Contains L-glutamine, but no sodium bicarbonate.

F-12K Nutrient Mixture, Kaighn’s Modification (1X), liquidRecommended storage conditions: 2°C to 8°C.For product formulation please refer to the tables at the end of this chapter.


500 ml 21127-022



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Glasgow Minimum Essential MediumGlasgow Minimum Essential Medium was formulated to support the growth of BHK-21 cells.For product formulation please refer to the tables at the end of this chapter.

Glasgow Minimum Essential Medium (G-MEM, BHK-21) (1X), liquidRecommended storage conditions: 2°C to 8°C.Intended use: In vitro diagnostic (IVD).


500 ml 21710-025

Contains L-glutamine, but no tryptose phosphate broth.

Grace’s Insect Cell Culture MediaFor product formulation please refer to the tables at the end of this chapter.

Grace’s Insect Cell Culture Medium, supplemented (1X), liquidInsect cell culture tested.Recommended storage conditions: 2°C to 8°C, in the dark.


500 ml 11605-045

10 × 500 ml (case) 11605-086

Contains 3330 mg/L lactalbumin hydrolysate, 3330 mg/L yeastolate, and L-glutamine, but no insect hemolymph.

Grace’s Insect Cell Culture Medium, supplemented (2X), liquidRecommended storage conditions: 2°C to 8°C, in the dark.


100 ml 11667-037

Contains lactalbumin hydrolysate, yeastolate, and L-glutamine.

Grace’s Insect Cell Culture Medium, unsupplemented (1X), liquidInsect cell culture tested.Recommended storage conditions: 2°C to 8°C, in the dark.


500 ml 11595-030

Contains L-glutamine, but no L-methionine or insect hemolymph.

Grace’s Insect Cell Culture Medium, powderInsect cell culture tested.Recommended storage conditions: 2°C to 8°C, in the dark.


10 × 1 L 11300-027


Related products PageInsect cell expression products . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 98Sf9 cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .175


36


Improved MEM Zn++ Option (Richter’s Modification)For product formulation please refer to the tables at the end of this chapter.

Improved MEM Zn++ Option (Richter’s Modification), liquidRecommended storage conditions: 2°C to 8°C.


500 ml 10373-017

Contains L-glutamine, 2 mg/L L-proline, and 50 µg/ml gentamicin sulfate, but no insulin, HEPES buffer, or phenol red.

IPL-41 Insect MediumFor product formulation please refer to the tables at the end of this chapter.

IPL-41 Insect Medium (1X), liquidInsect cell culture tested.Recommended storage conditions: 2°C to 8°C, in the dark.


500 ml 11405-057

Contains L-glutamine, calcium chloride, and sodium bicarbonate, but no tryptose phosphate broth.

Iscove’s Modified Dulbecco’s MediaIscove’s Modified Dulbecco’s Media are highly enriched synthetic media. They are well suited for rapidly proliferating, high-density cell cultures.For product formulation please refer to the tables at the end of this chapter.

Iscove’s Modified Dulbecco’s Medium (IMDM) (1X), liquidRecommended storage conditions: 2°C to 8°C.Intended use: In vitro diagnostic (IVD).


500 ml 21980-032

10 × 500 ml (case) 21980-065

1,000 ml 12440-046

Contains L-glutamine, 25 mM HEPES buffer, and 3,024 mg/L sodium bicarbonate, but no α-thioglycerol or 2-mercaptoethanol.

Quantity Cat. no.

500 ml 31980-022

10 × 500 ml (case) 31980-048

A GlutaMAX™-I medium. Contains GlutaMAX™-I substituted on a molar equivalent basis for L-glutamine, 25 mM HEPES buffer, and 3,024 mg/L sodium bicarbonate.

Quantity Cat. no.

500 ml 21056-023

Contains L-glutamine, 25 mM HEPES buffer, and 3,024 mg/L sodium bicarbonate, but no α-thioglycerol, 2-mercaptoethanol, or phenol red.

Iscove’s Modified Dulbecco’s Medium (IMDM), powderRecommended storage conditions: 2°C to 8°C.Intended use: In vitro diagnostic (IVD).


1 L 42200-014

10 L 42200-030

Contains L-glutamine and 25 mM HEPES buffer, but no sodium bicarbonate, α-thioglycerol, or 2-mercaptoethanol.

Related products PageInsect cell expression products . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 98


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McCoy’s 5A Medium (Modified)McCoy’s Medium (modified) was formulated for the propagation of human lympho cytes. It was based on the original formulation of McCoy as modified by Hsu and Kellog and further modified by Iwakata and Grace.For product formulation please refer to the tables at the end of this chapter.

McCoy’s 5A Medium (modified) (1X), liquidRecommended storage conditions: 2°C to 8°C.Intended use: In vitro diagnostic (IVD).


500 ml 26600-023

10 × 500 ml (case) 26600-080


Quantity Cat. no.

500 ml 22330-021

Contains L-glutamine and 25 mM HEPES buffer.

MCDB 131 MediumMCDB 131 was developed by Knedler and Ham as a reduced serum–supple-mented medium for the culture of human micro vascular endo thelial cells. This formulation has been reported to support clonal growth of human omental microvascular cells when supplemented with 0.7% dialyzed FBS, 10 ng/ml EGF, and 1 µg/ml hydrocortisone.For product formulation please refer to the tables at the end of this chapter.

ReferenceKnedler and Ham (1987) In Vitro Cell. Develop. Biol. 23, 481.

MCDB 131 Medium (1X), liquidRecommended storage conditions: 2°C to 8°C.


500 ml 10372-019


Leibovitz’s L-15 MediaLeibovitz ’s L-15 Media are suited for supporting cell growth in non–CO2-equilibrated environments. These formulations were developed without a sodium bicarbonate buffer system.For product formulation please refer to the tables at the end of this chapter.

Leibovitz’s L-15 Medium (1X), liquidRecommended storage conditions: 2°C to 8°C.Intended use: In vitro diagnostic (IVD).


500 ml 11415-049

10 × 500 ml (case) 11415-056


Recommended storage conditions: 2°C to 8°C.Quantity Cat. no.

500 ml 21083-027

Contains L-glutamine, but no phenol red.

Leibovitz’s L-15 Medium, powderRecommended storage conditions: 2°C to 8°C.Intended use: In vitro diagnostic (IVD).


1 L 41300-021

10 L 41300-070



38


Media 199Media 199 were formulated for nutritional studies of chick embryo fibroblasts. Media 199 are formulated with either Earle’s salts or Hanks’ salts. Media utilizing Earle’s salts are buffered with a bicarbonate/carbonic acid system and will maintain pH in a CO2 incubator. Use of Earle’s salts in atmospheric conditions will result in rapid rise in pH of the culture medium. Media utilizing Hanks’ salts are buffered with salt solutions designed for atmospheric equilibration, and use in a CO2 incubator will result in a rapid drop in pH of the culture medium.For product formulation please refer to the tables at the end of this chapter.

Medium 199 with Earle’s Salts

Medium 199 (1X), liquidRecommended storage conditions: 2°C to 8°C.Intended use: In vitro diagnostic (IVD).


500 ml 31150-022

10 × 500 ml (case) 31150-030

Contains L-glutamine and 2,200 mg/L sodium bicarbonate.

Quantity Cat. no.

500 ml 22340-020

Contains 2,200 mg/L sodium bicarbonate, L-glutamine, and 25 mM HEPES buffer.

Quantity Cat. no.

500 ml 11043-023

Contains L-glutamine and 2,200 mg/L sodium bicarbonate, but no phenol red.



500 ml 21180-021

Contains L-glutamine but no sodium bicarbonate.

Medium 199, powderRecommended storage conditions: 2°C to 8°C.Intended use: In vitro diagnostic (IVD).


1 L 31100-027

1 × 50 L 31100-019

Contains Earle’s salts and L-glutamine, but no sodium bicarbonate.

Medium 199 with Hanks’ Salts



500 ml 22350-029



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Minimum Essential Media (MEM)Minimum Essential Media are patterned after Eagle’s Media and are well suited for the growth of a broad spectrum of mammalian cells.

Comparison of Minimum Essential Media (MEM) with Earle’s SaltsBase cat. no.

With L-glutamine 31095* 22571 † 22561 † 41061 † 61100* 41500 11900 † 12000 †

With non-essential amino acids ● ● ● ● ● ●

With ribonucleosides and deoxyribonucleosides ● ● ●


α-modification ● ● ● ● ●

Powder § ● ● ● ●

Without L-glutamine 21090* 10370 11380 32360 51200 41090 32561 † 32571 † 21430 11700


With non-essential amino acids ● ● ●

With GlutaMAX™-I ‡ ● ● ●

With ribonucleosides and deoxyribonucleosides ●


α-modification ● ●

S-MEM modified for suspension culture (Ca2+ free) ●

10X powder § ●

* Standard formulation, as published in Eagle, H. (1959) Science 130, 432.

† MEM-α glutamine dipeptide, which can be used in the place of L-glutamine in culture media. It is substituted on an equimolar basis for L-glutamine.

§ Powdered media and concentrated formulations do not contain sodium bicarbonate.

‡ GlutaMAX™-I supplement is L-alanyl-L-glutamine, a glutamine dipeptide, which can be used in place of L-glutamine in culture media. It is substituted on an equimolar basis for L-glutamine.




40


Advanced Minimum Essential Medium (MEM) (1X), liquid (with L-glutamine)Recommended storage conditions: 2°C to 8°C, in the dark.For product formulation please refer to the tables at the end of this chapter. Additional information is available at www.invitrogen.com/advanced.

ReferencePrice, P. and Staines, D. (2004) Advanced Media: Reduced Sera Solutions, Quest 3, 25–30.


500 ml 12492-013

10 × 500 ml (case) 12492-021

Contains 110 mg/L of sodium pyruvate, non-essential amino acids (NEAA), but no L-glutamine.

Minimum Essential Medium (MEM) with Earle’s Salts (with L-glutamine)

Minimum Essential Medium (MEM) (1X), liquidRecommended storage conditions: 2°C to 8°C.Intended use: In vitro diagnostic (IVD).For product formulation please refer to the tables at the end of this chapter.


500 ml 31095-029

10 × 500 ml (case) 31095-052

Contains Earle’s salts and L-glutamine.

Minimum Essential Medium (MEM) α Medium (1X), liquidRecommended storage conditions: 2°C to 8°C.Intended use: In vitro diagnostic (IVD).For product formulation please refer to the tables at the end of this chapter.


500 ml 22571-020

10 × 500 ml (case) 22571-038

Contains L-glutamine, ribonucleosides, and deoxyribonucleosides.

Quantity Cat. no.

500 ml 22561-021

10 × 500 ml (case) 22561-054

Contains L-glutamine, but no ribonucleosides or deoxyribonucleosides.

Quantity Cat. no.

500 ml 41061-029

Contains L-glutamine, ribonucleosides, and deoxyribonucleosides, but no phenol red.

Minimum Essential Medium (MEM), powderRecommended storage conditions: 2°C to 8°C.Intended use: In vitro diagnostic (IVD).For product formulation please refer to the tables at the end of this chapter.


1 × 10 L 61100-087

1 × 50 L 61100-103

Contains Earle’s salts and L-glutamine, but no sodium bicarbonate.

Quantity Cat. no.

1 × 10 L 41500-018

1 × 50 L 41500-083

Contains Earle’s salts, L-glutamine, and non-essential amino acids, but no sodium bicarbonate.

Minimum Essential Medium (MEM) α Medium, powderRecommended storage conditions: 2°C to 8°C.Intended use: In vitro diagnostic (IVD).For product formulation please refer to the tables at the end of this chapter.


1 × 10 L 11900-073

Contains L-glutamine, ribonucleosides, and deoxyribonucleosides, but no sodium bicarbonate.

Quantity Cat. no.

1 × 10 L 12000-063

Contains L-glutamine, but no ribonucleosides, deoxyribonucleosides, or sodium bicarbonate.


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Minimum Essential Medium (MEM) with Earle’s Salts (without L-glutamine)



500 ml 21090-022

10 × 500 ml (case) 21090-055


Quantity Cat. no.

500 ml 10370-047

10 × 500 ml (case) 10370-070

Contains non-essential amino acids, but no L-glutamine.

Quantity Cat. no.

500 ml 32360-026

Contains 25 mM HEPES buffer, but no L-glutamine.

Quantity Cat. no.

500 ml 51200-046

Contains no L-glutamine or phenol red.

Quantity Cat. no.

500 ml 41090-028

10 × 500 ml (case) 41090-093


Quantity Cat. no.

500 ml 42360-024

10 × 500 ml (case) 42360-081

A GlutaMAX™-I medium. Contains GlutaMAX™-I substituted on a molar equivalent basis for L-glutamine and 25 mM HEPES buffer.



500 ml 21430-020

Contains no L-glutamine or sodium bicarbonate.

Minimum Essential Medium (MEM) α Medium (1X), liquidRecommended storage conditions: 2°C to 8°C.Intended use: In vitro diagnostic (IVD).For product formulation please refer to the tables at the end of this chapter.


500 ml 32561-029

10 × 500 ml (case) 32561-094

A GlutaMAX™-I medium. Contains GlutaMAX™-I substituted on a molar equivalent basis for L-glutamine, but no ribonucleosides or deoxyribonucleosides.

Quantity Cat. no.

500 ml 32571-028

10 × 500 ml (case) 32571-093

A GlutaMAX™-I medium. Contains GlutaMAX™-I substituted on a molar equivalent basis for L-glutamine, ribonucleosides, and deoxyribonucleosides.


42


Minimum Essential Medium (MEM) with Hanks' Salts, liquid (with L-glutamine)Recommended storage conditions: 2°C to 8°C.Intended use: In vitro diagnostic (IVD).For product formulation please refer to the tables at the end of this chapter.


500 ml 21575-022

Modified Eagle MediaModified Eagle Medium (MEM) (2X), liquidRecommended storage conditions: 2°C to 8°C.Intended use: In vitro diagnostic (IVD). For product formulation please refer to the tables at the end of this chapter.


500 ml 21935-028

Medium NCTC-109NCTC-109 is a synthetic medium that is suitable for general use in gen-erating and maintaining hybridoma cells. This medium typically requires the addition of a growth factor cocktail or serum.For product formulation please refer to the tables at the end of this chapter.

Medium NCTC-109 (1X), liquidRecommended storage conditions: 2°C to 8°C.Intended use: In vitro diagnostic (IVD).


500 ml 21340-054


Related products PageHybridoma and monoclonal antibody products . . . . . . . . . . . . . . . . . . . . .106

F-10 Nutrient MixturesF-10 Nutrient Mixture (Ham) was formulated for serum-free growth of Chinese hamster ovary (CHO) cells and growth of various mammalian cell lines with serum supplementation.For ordering information, please refer to page 34.

F-12 Nutrient MixturesF-12 Nutrient Mixture was formulated for single-cell plating of near-diploid Chinese hamster ovary (CHO) cells. For ordering information, please refer to page 34.


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Opti-MEM® I Reduced-Serum MediaOpti-MEM® I is a modification of Eagle’s Minimum Essential Medium, buffered with HEPES and sodium bicarbonate (2.4 g/L) and supplemented with hypoxanthine, thymidine, sodium pyruvate, L-glutamine, trace elements, and growth factors. The concentration of phenol red has been reduced to 1.1 mg/L. Most cells routinely cultured in serum-supplemented medium may be transferred directly into Opti-MEM® I with a minimum of 50% reduction in serum. Further significant reductions in serum requirements have been achieved with myelomas (Sp2/O-Ag14, P3×63 Ag8.653, P3-NSI-Ag4-1) and derived hybridomas, as well as with fibroblasts and epithelial cells of both normal and tumor origins. For very low serum supplementation (<1%) with attachment-dependent cells, add 500 to 1,000 mg/L calcium chloride. Ideal for use during cati onic lipid transfections.1

Reference1. FOCUS on Applications, Opti-MEM® I Reduced-Serum Medium: Application in Transfection, Technical Bulletin.

Opti-MEM® I Reduced-Serum Medium (1X), liquidRecommended storage conditions: 2°C to 8°C, in the dark.Intended use: In vitro diagnostic (IVD).


100 ml 31985-062

500 ml 31985-047

10 × 500 ml (case) 31985-054

Contains HEPES buffer, 2,400 mg/L sodium bicarbonate, hypoxanthine, thymidine, sodium pyruvate, L-glutamine, trace elements, growth factors, and phenol red reduced to 1.1 mg/L.

Opti-MEM® I Reduced-Serum Medium (1X), liquid (without phenol red)Recommended storage conditions: 2°C to 8°C, in the dark.


500 ml 11058-021

Contains HEPES buffer, 2,400 mg/L sodium bicarbonate, hypoxanthine, thymidine, sodium pyruvate, L-glutamine, trace elements, and growth factors, but no phenol red.

Opti-MEM® I Reduced-Serum Medium (1X), liquid (with GlutaMAX™-I)Recommended storage conditions: 2°C to 8°C, in the dark.Intended use: In vitro diagnostic (IVD).


500 ml 51985-026

10 × 500 ml (case) 51985-042

A GlutaMAX™-I medium. Contains GlutaMAX™-I substituted on a molar equivalent basis for L-glutamine, HEPES buffer, 2,400 mg/L sodium bicarbonate, hypoxanthine, thymidine, sodium pyruvate, trace elements, growth factors, and phenol red reduced to 1.1 mg/L.

Opti-MEM® I Reduced-Serum Medium, powderRecommended storage conditions: 2°C to 8°C, in the dark.Intended use: In vitro diagnostic (IVD).Note: See page 84 for procedure to prepare powdered Opti-MEM® I.


1 × 10 L 22600-134

A modification of MEM (Eagle’s). Contains HEPES buffer, hypo xanthine, thymidine, sodium pyruvate, L-glutamine, trace elements, growth factors, and phenol red reduced to 1.1 mg/L, but no sodium bicarbonate.

Lipofectamine™ 2000 Transfection ReagentLipofectamine™ 2000 Transfection Reagent is a proprietary cationic lipid formulation that offers the highest transfection efficiencies on the widest variety of cell lines with high protein expression levels or gene knockdown activity (RNAi). It is the leading transfection reagent for simple, effective, and optimal nucleic acid delivery in a wide range of eukaryotic cell types and is proven to work in high-throughput applications.Recommended storage conditions: 2°C to 8°C.

Ordering InformationQuantity Cat. no.0.75 ml 11668-027

1.5 ml 11668-019


44


RPMI Media 1640RPMI Media 1640 are enriched formulations with extensive applications for mammalian cells. They were formulated for suspension cultures or monolayer culture of human leukemic cells.

RPMI Media 1640 Comparison ChartBase cat. no.

With L-glutamine 21875* 11835 11879 52400 51800* 13018 27016

With HEPES buffer (25 mM) ● ●

Without glucose ●


Without folate ●

Powder † ● ●

Without L-glutamine 31870* 61870 72400

With GlutaMAX™-I § ● ●


* Standard formulation, as published in Moore, G.E., Gerner, R.E., and Franklin, H.A. (1967) JAMA 199, 519. † Powdered media do not contain sodium bicarbonate. § GlutaMAX™-I supplement is L-alanyl-L-glutamine, a glutamine dipeptide, which can be used in the place of L-glutamine in culture media. It is substituted on an equimolar basis for L-glutamine.


For the exact composition of each product, please refer to the formulation tables on page 107.


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Advanced RPMI Medium 1640 (1X), liquidRecommended storage conditions: 2°C to 8°C, darkwww.invitrogen.com/advanced

Reference Price, P. and Staines, D. (2004) Advanced Media: Reduced Sera Solutions, Quest 3, 25–30.


500 ml 12633-012

10 × 500 ml (case) 12633-020

Contains 2,000 mg/L D-glucose, non-essential amino acids (NEAA), and 110 mg/L sodium pyruvate, but no L-glutamine.

RPMI Medium 1640 (1X), liquidRecommended storage conditions: 2°C to 8°C.Intended use: In vitro diagnostic (IVD).For product formulation please refer to the tables at the end of this chapter.


100 ml 21875-042

500 ml 21875-034

10 × 500 ml (case) 21875-091

6 × 1,000 ml (case) 21875-158


Quantity Cat. no.

500 ml 11835-063

Contains L-glutamine, but no phenol red.

Quantity Cat. no.

100 ml 52400-017

500 ml 52400-025

10 × 500 ml (case) 52400-041

Contains L-glutamine and 25 mM HEPES buffer.

Quantity Cat. no.

500 ml 11879-020

Contains L-glutamine, but no glucose.

Quantity Cat. no.

500 ml 27016-021

Contains L-glutamine, but no folic acid.

Quantity Cat. no.

500 ml 31870-025

10 × 500 ml (case) 31870-074


Quantity Cat. no.

500 ml 61870-010

10 × 500 ml (case) 61870-044


Quantity Cat. no.

500 ml 72400-021

10 × 500 ml (case) 72400-054

A GlutaMAX™-I medium. Contains GlutaMAX™-I substituted on a molar equivalent basis for L-glutamine and 25 mM HEPES buffer.

RPMI Medium 1640, powderRecommended storage conditions: 2°C to 8°C.Intended use: In vitro diagnostic (IVD).For product formulation please refer to the tables at the end of this chapter.


1 L 51800-019

1 × 10 L 51800-035

1 × 50 L 51800-043


Quantity Cat. no.

1 L 13018-015

1 × 10 L 13018-031

Contains L-glutamine and 25 mM HEPES buffer, but no sodium bicarbonate.


46


Schneider’s Drosophila MediumFor product formulation please refer to the tables at the end of this chapter.

Schneider’s Drosophila Medium (1X), liquidModified and insect cell culture tested.Recommended storage conditions: 2°C to 8°C.


500 ml 21720-024



Waymouth’s MB 752/1 MediumWaymouth’s MB 752/1 Media was formulated for studies on the nutrition, metabolism, and growth of Strain L sublines, NCTC clone 929.For product formulation please refer to the tables at the end of this chapter.

Waymouth’s MB 752/1 Medium (1X), liquidRecommended storage conditions: 2°C to 8°C.Intended use: In vitro diagnostic (IVD).


500 ml 31220-023


Williams’ Medium EWilliams’ Medium E was formulated for long-term cell cultures of adult rat liver epithelial cells.For product formulation please refer to the tables at the end of this chapter.

Williams’ Medium E (1X), liquidRecommended storage conditions: 2°C to 8°C.Intended use: In vitro diagnostic (IVD).


500 ml 22551-022



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Media Preparation

Liquid Media: Preparing 1X Solutions from 10X ConcentratesTo prepare an acceptable final 1X solution, perform the following procedures under aseptic conditions. We recommend distilled water (base cat. no. 15230) and 7.5% Sodium Bicarbonate Solution (base cat. no. 25080) for use in this protocol. Use sterile solutions of 1 N sodium hydroxide or hydrochloric acid prepared by membrane filtration using appropriate filters. Also note that because we adjust the pH of all 10X media and 10X balanced salt solution concentrates for solubility, you may need to adjust the pH after dilution and add sodium bicarbonate, as appropriate.1. Aseptically dilute 100 ml of 10X concentrate with approximately 850 ml of distilled water.2. Aseptically add the correct amount of 7.5% sodium bicarbonate solution (see table below for concentrations). 3. Adjust the pH as necessary with 1 N hydrochloric acid or 1 N sodium hydroxide (see table below for 1X pH values).4. QS to volume with distilled water.5. Dispense the solution into sterile containers. Cap the bottles tightly with sterile closures and store them at the recommended temperature (see

product descriptions in this chapter).

Procedure 1: Preparing powdered media1. To a mixing container that is as close to the final volume as

possible, add 2–3% less distilled water than the desired total volume of medium. For example, for 1 L of medium start with 970 ml to 980 ml of distilled water.

2. Add powdered medium to room temperature (20°C to 30°C) water with gentle stirring. Do not heat the water.

3. Rinse the inside of the package to remove all traces of powder.4. Add NaHCO3 (see table below for quantity) to medium. See the table

on the next page for sodium bicarbonate concentration.5. Adjust the pH to 0.2 and 0.3 units below the desired final working pH

by slowly adding, with stirring, either 1 N NaOH or 1 N HCl. The pH may rise 0.1 to 0.3 units upon filtration.

6. Dilute the medium to the desired volume with distilled water. Do not overmix. Keep the container closed until the medium is filtered.

7. Process the medium immediately into sterile containers by membrane filtration using a 0.2 µm filter. We recommend using a positive-pressure system.

Procedure 2: Preparing powdered Leibovitz’s L-15 Media1. To a mixing container that is as close to the final volume as possible, add

2–3% less distilled water than the desired total volume of medium.2. Add powdered medium to room temperature (20°C to 30°C) water

with gentle stirring. Do not heat the water.3. Rinse the inside of the package to remove all traces of powder.4. Adjust the pH to 7.6 by slowly adding, with stirring, 1 N NaOH or 1 N

HCl.5. Dilute the liquid to the desired volume with distilled water and stir

until dissolved. Do not overmix.6. Process the medium immediately into sterile containers by

membrane filtration using a 0.2 µm filter. We recommend using a positive-pressure system.

Sodium Bicarbonate Concentrations Achieved Using 7.5% Sodium Bicarbonate Solution (base cat. no. 25080)NaHCO3

pH of 10X solution Final concentration (g/L) 7.5% solution to add (ml/L)pH range after 1:10 dilution

and addition of NaHCO3 Recommended pH 1X solution*

Base cat. no. Balanced salt solutions

14065 5.8–6.1 0.35 4.7 7.5–7.8 7.0–7.4

14080 † 4.4–4.7 – – 5.2–5.5 7.0–7.2

14185 5.8–6.1 0.35 4.7 6.0–6.3 7.0–7.4

14200 † 6.7–7.0 – – 7.1–7.4 7.0–7.2

Liquid media

21430 4.9–5.4 2.2 29.3 7.6–7.9 7.0–7.4

* pH range will vary depending on pH of water used for dilution. † Formulation does not require addition of NaHCO3.


48


Procedure 3: Preparing powdered Iscove’s Modified Dulbecco’s Media1. To a mixing container that is as close to the final volume as possible, add

2–3% less distilled water than the desired total volume of medium.2. Add powdered medium to room temperature (20°C to 30°C) water

with gentle stirring. Do not heat the water.3. Rinse the inside of the package to remove all traces of powder.4. Add 3.024 g of NaHCO3/L of medium.5. Dilute the medium to the desired volume with distilled water and stir

until dissolved. Do not overmix.6. Do not adjust the pH. Keep the container closed until the medium is

filtered.7. Process the medium immediately into sterile containers by membrane

filtration using a 0.2 µm filter. We recommend using a positive-pressure system.

Procedure 4: Preparing powdered Opti-MEM® I Reduced-Serum Medium1. To a mixing container that is as close to the final volume as

possible, add 2–3% less distilled water than the desired total volume of medium. For example, for 1 L of medium start with 970 ml to 980 ml of distilled water.


3. Rinse the inside of the package to remove all traces of powder.4. Stir until completely in solution (usually within 10 min).5. Adjust the pH to between 0.2 and 0.3 units below the desired final pH by

slowly adding, with stirring, either 1 N NaOH or 1 N HCl (recommended final pH is 7.3 ± 0.1). The pH may rise 0.1 to 0.3 units upon filtration.

6. Add 2.4 g of reagent-grade NaHCO3 and, if appropriate, 1.0 ml of 5.5 × 10–2 M 2-mercaptoethanol (1,000X)/L of medium.

7. Dilute the medium to the desired volume with distilled water and stir until dissolved. Do not overmix. Keep the container closed until the medium is filtered.

8. Process the medium immediately into sterile containers by membrane filtration using a 0.2 µm filter. We recommend using a positive-pressure system. Low–protein-adsorbent filters are required.

Procedure 5: Preparing powdered RPMI 1640 Media1. To a mixing container that is as close to the final volume as pos-

sible, add 2–3% less distilled water than the desired total volume of medium. For example, for 1 L of medium start with 970 ml to 980 ml of distilled water.


3. Rinse the inside of the package to remove all traces of powder.4. Add 2.0 g of NaHCO3/L of medium (it will appear cloudy because of

incomplete solubilization).5. Adjust the pH to between 0.2 and 0.3 below the desired final working

pH by slowly adding, with stirring, either 1 N NaOH or 1 N HCl. The pH may rise 0.1 to 0.3 units upon filtration.

6. Dilute the medium to the desired volume with distilled water and stir until dissolved. Do not overmix. Keep the container closed until the medium is filtered.

7. Process the medium immediately into sterile containers by membrane filtration using a 0.2 µm filter. We recommend using a positive-pressure system.

Preparation of Powdered MediaNaHCO3

Procedure number* g/L

ml/L 7.5% solution

Recommended working pH Base cat. no.

1 0.35 4.7 6.0–6.2 11300

1 2.2 29.3 7.0–7.4 11900

1 2.2 29.3 7.0–7.4 12000

1 3.7 49.3 7.0–7.4 52100

3 3.02 40.3 (6.9–7.3) 7.0-

7.4

42200

1 (1.2) 1,125

(16.0) 15 7.1–7.4 42400

1 2.438 (32.5) 15 7.1–7.4 32500

1 3.7 49.3 7.0–7.4 12800

1 0 0 7.0–7.2 21600

1 1.18 15.7 7.6–7.9 21700

4 2.4 32.0 7.2–7.4 22600

5 2.0 26.7 7.0–7.4 22340

1 2.2 29.3 (7.1–7.4) 7.0-

7.4

31100

1 3.7 49.3 7.0–7.4 31600

5 2.0 26.7 7.0–7.4 51800

2 † † 7.3–7.6 41300

1 2.2 29.3 7.0–7.4 41500

1 2.2 29.3 7.0–7.4 61100

* See procedures 1 through 5. † Formulations do not require addition of NaHCO3.


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E-Z Hold™ Plastic BottlesGIBCO® E-Z Hold™ Bottles were designed for cell culturists from polyethylene terephthalate (PET) plastic, the most recycled plastic in the world. These bottles are lightweight and virtually unbreakable.

Bottle features include a wide mouth and no-drip spout for easy pipetting, two flat sides for easy gripping, and an overall round shape to facilitate swirling of contents in the bottle.

This product is the subject of US Patent 5,246,127.


100 ml E-Z Hold™ bottles with caps 264 units/case 10833-010

500 ml E-Z Hold™ bottles with caps 66 units/case 10833-028

1,000 ml E-Z Hold™ bottles with caps 24 units/case 10833-036

Stak Pack™ AccessoriesStak Pack™ accessories are for use with the GIBCO® Stak Pack™ delivery system. The contents of each package are radiation sterilized and are suitable for aseptic cell culture procedures unless the package has been opened or damaged.

This product is the subject of US Patent 4,869,398.


36" transfer tube with male luer lock; radiation sterilized 1 12002-010

36" transfer tube with female luer lock; radiation sterilized 1 10265-015

8" interconnecting tube; radiation sterilized 1 11003-019


50


Basal Medium Eagle*Base cat. no. 41010

Component 1X liquid, mg/L

Inorganic salts

CaCl2 (anhyd.) 264.00

KCl 400.00

MgSO4 (anhyd.) 200.00

NaCl 6800.00

NaHCO3 2200.00

NaH2PO4•H2O † 158.00

Other components

D-Glucose 1000.00

Phenol red 10.00

Amino acids

L-Arginine • HCl 21.00

L-Cystine • 2HCl 12.00

L-Histidine 8.00

L-Isoleucine 26.00

L-Leucine 26.00

L-Lysine • HCl 29.00

L-Methionine 7.50

L-Phenylalanine 16.50

L-Threonine 24.00

L-Tryptophan 4.00

L-Tyrosine • 2Na • 2H2O

18.00

L-Valine 23.50

Vitamins

Biotin 1.00

D-Ca pantothenate 1.00

Choline chloride 1.00

Folic acid 1.00

i-Inositol 1.80

Niacinamide 1.00

Pyridoxal HCl 1.00

Riboflavin 0.10

Thiamine HCl 1.00

Eagle, H (1965) Proc. Soc . Exp. Biol. Med. 89. 362*also known as Nicotinamide

Media FormulationsCMRL 1066 Medium 1

Base cat. no. 21530 21540


10X liquid, mg/L

Inorganic salts

CaCl2 (anhyd.) 264.00 2000.00

KCl 400.00 4000.00

MgSO4 .7H2O 200.00 2000.00

NaCl 6799.00 67990.00

NaHCO3 2200.00 -

NaH2PO4 • H2O 158.00 1580.00

Other components

Cocarboxylase 1.00 10.00

Coenzyme A 2.50 25.00

2-Deoxyadenosine 10.00 100.00

2-Deoxycytidine • HCl 10.00 100.00

2-Deoxyguanosine 10.00 100.00

Diphosphopyridine nucleotide

7.00 70.00

Ethanol for solubilsing lipid components

16.0 160.00

Flavin adenine dinucleotide

1.00 10.00

D-Glucose 1000.00 10000.00

Glutathione (reduced) 10.00 100.00

5-Methyl-deoxycytidine 0.10 1.00

Phenol red* 20.00 200.00

Sodium acetate • 3H2O 83.00 830.00

Sodium glucuronate • H2O

4.00 40.00

Thymidine 10.00 100.00

Triphosphopyridine nucleotide

1.00 10.00

Tween 80 5.00 50.00

Uridine triphosphate 1.00 10.00

Amino acids

L-Alanine 25.00 250.00

L-Arginine HCl 70.00 700.00

L-Aspartic acid 30.00 300.00

L-Cystine (free base) 199.88 1998.80

L-Cystine • 2HCl 20.00 200.00

L-Glutamic acid 75.00 750.00

Glycine 50.00 500.00

L-Histidine HCl • H2O 20.00 200.00

Hydroxy-L-proline 10.00 100.00

L-Isoleucine 20.00 200.00

L-Leucine 60.00 600.00

L-Lysine • HCl 70.00 700.00

L-Methionine 15.00 150.00

L-Phenylalanine 25.00 250.00

L-Proline 40.00 400.00

L-Serine 25.00 250.00

L-Threonine 30.00 300.00

L-Tryptophan 10.00 100.00

L-Tyrosine 40.00 400.00

L-Valine 25.00 25.00

CMRL 1066 Medium 1

Base cat. no.


10Xliquidmg/L

Vitamins

Ascorbic acid 50.00 500.00

D-Biotin 0.01 0.10

D-Ca pantothenate 0.01 0.10

Cholesterol 0.20 2.00

Choline chloride 0.50 5.00

Folic acid 0.01 0.10

i-Inositol 0.05 0.50

Niacin 0.025 0.25

Niacinamide 0.025 0.25

Para-aminobenzoic acid 0.05 0.5

Pyridoxine HCl 0.025 0.254

Riboflavin 0.01 0.1

Thiamine HCl 0.01 0.10

1. Parker R. C, et al (1957) Special publica-tions, N.Y. Academy of Sciences, 5, 303a. Values established by the Tissue Culture Association Standards Committee.Tween 80 is a registered trademark of I.CI Americas, Inc *also known as Nicotinamide


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Brinster ’s BMOC-3 MediumBase cat. no. 11126


Inorganic salts

NaCl 5546.00

KCl 356.00

CaCl2 189.00

KH2PO4 162.00

MgSO4 • 7H2O 294.00

Glucose 1000.00

Sodium bicarbonate

2106.00

DL-Lactic acid Na 2.773

Sodium pyruvate 56.00

Bovine albumin fraction V 5000.00

Riboflavin 0.04

Thiamine HCI 0.30

Vitamin B12 1.40

1. Ham, RG. (1965) Proc. Nat. Arad.Sci., 53, 288.2. Kaighn, M. E., Tissue Culture Methods and Applications, (1973), Kruse, P.F., and Patterson Jr M.D. ed p.54-58*also known as Nicotinamide

F-12 Nutrient Mixture (Ham) Kaighn's Modification1.2

Base cat. no. 21127

Component Powder, mg/LInorganic salts

CaCl2 (anhyd.) 102.00

KH4 • PO4 59.00

CuSO4 • 5H2O 0.002

FeSO4 • 7H2O 0.80

KCI 285.00

MgCI2 (anhyd.) 49.70

MgSO4 192.00

NaCI 7530.00

NaHCO3 2500.00

Na2 HPO4 (anhyd) 115.50

ZnSO4 • 7H2O 0.144

Other components

D-Glucose 1260.00

Hypoxanthine • Na 4.00

Lipoic Acid 0.21

Phenol Red 3.00

Putrescine 2HCI 0.32

Sodium Pyruvate 220.00

Thymidine 0.70

Amino acids

L-Alanine 18.00

L-Arginine • HCI 422.00

L-Asparagine • H2O 30.00

L-Aspartic Acid 26.00

L-Cystine • HCI • H2O 70.00

L-Glutamine Acid 29.00

L-Glutamine 292.00

Glycine 15.00

L-Histidine HCI • H2O 45.80

L-Isoleucine 7.88

L-Leucine 26.20

L-Lysine • HCI 73.00

L-Methionine 8.96


L-Proline 69.00

L-Serine 21.00

L-Threonine 23.00

L-Tryptophan 2.04

L-Tyrosine • 2 Na • 2H2O 13.50

L-Valine 23.00

Vitamins

Biotin 0.07

D-Calcium pantothenate 0.50

Choline Chloride 14.00

Folic Acid 1.30

i-Inositol 18.00

Niacinamide* 0.037

Pyridoxine HCI 0.06


52


Dulbecco’s Modified Eagle Media (DMEM)1

Base cat. no. 10938 11880 11960 11966 11971 12800 21013 21063 21068


1X liquid, mg/L

1X liquid, mg/L

1X liquid, mg/L

1X liquid, mg/L

Powder, mg/L

1X liquid, mg/L

1X liquid, mg/L

1X liquid, mg/L

Inorganic salts

CaCl2 • 2H2O 264.00 2644.00 – – – – – – –

CaCl2 (anhyd.) – – 200.00 200.00 200.00 200.00 200.00 200.00 –

Fe(NO3)3 • 9H2O 0.10 0.10 0.10 0.10 0.10 0.10 0.10 0.10 0.10

KCl 400.00 400.00 400.00 400.00 400.00 400.00 400.00 400.00 400.00

MgSO4 (anhyd.) – 97.67 97.67 97.67 97.67 97.67 97.67 97.67 97.67

MgSO4 • 7H2O 200.00 200.00 – – – – – – –

NaCl 6400.00 6400.00 6400.00 4750.00 4750.00 6,400.00 6400.00 4750.00 6400.00

NaHCO3 3700.00 3700.00 3700.00 3700.00 3700.00 - 3700.00 3700.00 3700.00

NaH2PO4 • 2H2O 141.00 141.00 – – – – – – –

NaH2PO4 • H2O* – – 125.00 125.00 – 125.00 125.00 125.00 125.00

Other components

D-Glucose 4500.00 1000.00 4500.00 – 4500.00 4,500.00 4,500.00 4500.00 4500.00

Phenol red 17.00 – 15.00 15.00 15.00 15.00 15.00 – 15.00

HEPES – – – – – – – 5958.00 –

Sodium pyruvate – 110.00 – – – 110.00 – – –

Amino acids

L-Alanine 35.60 – – – – – – – –

L-Aspargine 60.00 – – – – – – – –

L-Arginine • HCl 84.00 84.00 84.00 84.00 84.00 84.00 84.00 84.00 84.00

L-Apsrtic Acid 53.00 – – – – – – – –

L-Cystine 48.00 48.00 – – – – – – –

L-Cystine • 2HCl – – 63.00 63.00 63.00 63.00 – 63.00 63.00

L-Glutamic Acid 59.00 – – – – – – – –

L-Glutamine – – – 584.00 584.00 584.00 – 584.00 –

L-Alanyl-L-glutamine – – – – – – –

Glycyl-L-Glutamine – – – – – – – –

Glycine 30.00 30.00 30.00 30.00 30.00 30.00 30.00 30..00 30..00

L-Histidine HCl • H2O 42.00 42.00 42.00 42.00 42.00 42.00 42.00 42.00 42.00

Amino acids, continued

L-Isoleucine 105.00 105.00 105.00 105.00 105.00 105.00 105.00 105.00 105.00

L-Leucine 105.00 105.00 105.00 105.00 105.00 105.00 105.00 105.00 105.00

L-Lysine • HCl 146.00 146.00 146.00 146.00 146.00 146.00 146.00 146.00 146.00

L-Methionine 30.00 30.00 30.00 30.00 30.00 30.00 - 30.00 30.00

L-Phenylalanine 66.00 66.00 66.00 66.00 66.00 66.00 66.00 66.00 66.00

L-Serine 42.00 42.00 42.00 42.00 42.00 42.00 42.00 42.00 42.00

L-Threonine 95.00 95.00 95.00 95.00 95.00 95.00 95.00 95.00 95.00

L-Tryptophan 16.00 16.00 16.00 16.00 16.00 16.00 16.00 16.00 16.00

L-Tyrosine 72.00 72.00 – – – – – – –

L-Tyrosine • 2Na • 2H2O – – 104.00 104.00 104.00 104.00 104.00 104.00 104.00

L-Valine 94.00 94.00 94.00 94.00 94.00 94.00 94.00 94.00 94.00

Vitamins

D-Ca pantothenate 4.00 4.00 4.00 4.00 4.00 4.00 4.00 4.00 4.00

Choline chloride 4.00 4.00 4.00 4.00 4.00 4.00 4.00 4.00 4.00

Folic acid 4.00 4.00 4.00 4.00 4.00 4.00 4.00 4.00 4.00

i-Inositol 7.20 7.20 7.20 7.20 7.20 7.20 7.20 7.20 7.20

Niacinamide* 4.00 4.00 4.00 4.00 4.00 4.00 4.00 4.00 4.00

Pyridoxine HCl 4.00 4.00 4.00 4.00 4.00 – 4.00 4.00 4.00

Pyridoxal HCI – – – – – 4.00 – – –

Riboflavin 0.40 0.40 0.40 0.40 0.40 0.40 0.40 0.40 0.40

Thiamine HCl 4.00 4.00 4.00 4.00 4.00 4.00 4.00 4.00 4.00

1. Dulbecco, R., Freeman, G. (1959) Virology 8, 396, Smith, J. D,. Freeman, G,. Vogt, M. And Dulbecco, R. (1960) Virology 12, 185. Tissue Culture Association Standards Committee, In Vitro 6:2, 93.a. Values shown are in conformance with the Tissue Culture Association Standards Committee, In Vitro 6:2 93. *also known as Nicotinamide

–


edia3



continued


Base cat. no. 21885 21969 22320 31053 31600 31885 31965 31966 32430


1X liquid, mg/L

1X liquid, mg/L

1X liquid, mg/L

1X liquid, mg/L

1X liquid, mg/L

1X liquid, mg/L

1X liquid, mg/L

1X liquid, mg/L

Inorganic salts

CaCl2 • 2H2O 264.00 264.00 264.00 – – 264.00 264.00 264.00 264.00

CaCl2 (anhyd.) – – – 200.00 200.00 – – – –

Fe(NO3)3 • 9H2O 0.10 0.10 0.10 0.10 0.10 0.10 0.10 0.10 0.10

KCl 400.00 400.00 400.00 400.00 400.00 400.00 400.00 400.00 400.00

MgSO4 (anhyd.) – – – 97.67 97.67 – – – –

MgSO4 • 7H2O 200.00 200.00 200.00 – – 200.00 200.00 200.00 200.00

NaCl 6400.00 6400.00 3500.00 6400.00 6400.00 6,400.00 6400.00 6400.00 6400.00

NaHCO3 3700.00 3700.00 3700.00 3700.00 – 3700.00 3700.00 3700.00 3700.00

NaH2PO4 • 2H2O 141.00 141.00 141.00 – – 141.00 141.00 141.00 141.00

NaH2PO4 • H2O* – – – 125.00 125.00 – – – –

Other components

D-Glucose 1000.00 4500.00 1000.00 4500.00 1000.00 1000.00 4,500.00 4500.00 4500.00

Phenol red 15.00 15.00 15.00 – 15.00 15.00 15.00 15.00 15.00

HEPES – – 5958.00 – – – – – 5958.00

Sodium pyruvate 110.00 110.00 110.00 – 110.00 110.00 – 110.00 –

Amino acids

L-Alanine – – – – – – – – –

L-Asparagine – – – – – – – – –

L-Arginine • HCl 84.00 84.00 84.00 84.00 84.00 84.00 84.00 84.00 84.00

L-Cystine 48.00 48.00 48.00 – – 48.00 48.00 48.00 48.00

L-Cystine • 2HCl – – – 63.00 62.77 – – – –

L-Glutamine – – 580.00 – 584.00 580.00 – – –

L-Alanyl-L-glutamine 862.00 – – – – – – 862.00 862.00

Glycine 30.00 30.00 30.00 30.00 30.00 30.00 30.00 30.00 30.00

L-Histidine HCl • H2O 42.00 42.00 42.00 42.00 42.00 42.00 42.00 42.00 42.00

L-Isoleucine 105.00 105.00 105.00 105.00 105.00 105.00 105.00 105.00 105.00

L-Leucine 105.00 105.00 105.00 105.00 105.00 105.00 105.00 105.00 105.00

L-Lysine • HCl 146.00 146.00 146.00 146.00 146.00 146.00 146.00 146.00 146.00

L-Methionine 30.00 30.00 30.00 30.00 30.00 30.00 30.00 30.00 30.00

L-Phenylalanine 66.00 66.00 66.00 66.00 66.00 66.00 66.00 66.00 66.00

L-Proline – – – – – – - – –

L-Serine 42.00 42.00 42.00 42.00 42.00 42.00 42.00 42.00 42.00

L-Threonine 95.00 95.00 95.00 95.00 95.00 95.00 95.00 95.00 95.00

L-Tryptophan 16.00 16.00 16.00 16.00 16.00 16.00 16.00 16.00 16.00

L-Tyrosine 72.00 72.00 72.00 – – 72.00 72.00 72.00 72.00

L-Tyrosine • 2Na • 2H2O – – – 104.00 104.18 – – – –

L-Valine 94.00 94.00 94.00 94.00 94.00 94.00 94.00 94.00 94.00

Vitamins

D-Ca pantothenate 4.00 4.00 4.00 4.00 4.00 4.00 4.00 4.00 4.00

Choline chloride 4.00 4.00 4.00 4.00 4.00 4.00 4.00 4.00 4.00

Folic acid 4.00 4.00 4.00 4.00 4.00 4.00 4.00 4.00 4.00

i-Inositol 7.20 7.20 7.20 7.20 7.20 7.20 7.20 7.20 7.20

Niacinamide* 4.00 4.00 4.00 4.00 4.00 4.00 4.00 4.00 4.00

Pyridoxine HCI 4.00 – 4.00 4.00 4.00 4.00 4.00 4.00 4.00

Pyridoxal HCI – 4.00 – – – – – – –w

Riboflavin 0.40 0.40 0.40 0.40 0.40 0.40 0.40 0.40 0.40

Thiamine HCl 4.00 4.00 4.00 4.00 4.00 4.00 4.00 4.00 4.00


54



Base cat. no. 41965 41966 42430 52100 61965

Component1X liquid,

mg/L1X liquid,

mg/L1X liquid,

mg/L1X Powder,

mg/L1X liquid,

mg/L

Inorganic salts

CaCl2 • 2H2O 264.00 264.00 264.00 – 264.00

CaCl2 (anhyd.) – – – 200.00 –

Fe(NO3)3 • 9H2O 0.10 0.10 0.10 0.10 0.10

KCl 400.00 400.00 400.00 400.00 400.00

MgSO4 (anhyd.) – – – 97.67 –

MgSO4 • 7H2O 200.00 200.00 200.00 – 200.00

NaCl 6400.00 6400.00 3500.00 6400.00 6400.00

NaHCO3 3700.00 3700.00 3700.00 – 3700.00

NaH2PO4 • 2H2OO2 141.00 141.00 141.00 – 141.00

NaH2PO4 • H2Oa – – – 125.00 –

Other components

D-Glucose 4500.00 4500.00 4500.00 4500.00 4500.00

Phenol red 15.00 15.00 15.00 15.00 15.00

HEPES – – 5958.00 – –

Sodium pyruvate – 110.00 – – –

Amino acids

L-Arginine – – – – –

L-Asparagine – – – – –

L-Arginine • HCI 84.00 84.00 84.00 84.00 84.00

L-Aspartic acid – – – – –

L-Cystine 48.00 48.00 48.00 – 48.00

L-Cystine • 2HCl – – – 62.75 –

L-Glutamine acid – – – – –

L-Glutamine 580.00 580.00 580.00 580.00 580.00

L-Alanyl-L-Glutamine – – – – 862.00

Glycyl-L-Glutamine – – – – –

Glycine 30.00 30.00 30.00 30.00 30.00

L-Histidine HCl • H2O 42.00 42.00 42.00 42.00 42.00

L-Isoleucine 105.00 105.00 105.00 105.00 105.00

L-Leucine 105.00 105.00 105.00 105.00 105.00

L-Lysine • HCl 146.00 146.00 146.00 146.00 146.00

L-Methionine 30.00 30.00 30.00 30.00 30.00

L-Phenylalanine 66.00 66.00 66.00 66.00 66.00

L-Proline – – – – –

L-Serine 42.00 42.00 42.00 42.00 42.00

L-Threonine 95.00 95.00 95.00 95.00 95.00

L-Tryptophan 16.00 16.00 16.00 16.00 16.00

L-Tyrosine 72.00 72.00 72.00 – 72.00


L-Tyrosine (disodium salt) – – – 104.20 –

L-Valine 94.00 94.00 94.00 94.00 94.00

Vitamins

D-Ca pantothenate 4.00 4.00 4.00 4.00 4.00

Choline chloride 4.00 4.00 4.00 4.00 4.00

Folic acid 4.00 4.00 4.00 4.00 4.00


edia3




Base cat. no. 41965 41966 42430 52100 61965

Vitamins, continued

i-Inositol 7.20 7.20 7.20 7.20 7.20

Niacinamide* 4.00 4.00 4.00 4.00 4.00

Pyridoxine HCI 4.00 4.00 4.00 – 4.00

Pyridoxal HCI – – – 4.00 –

Riboflavin 0.40 0.40 0.40 0.40 0.40

Thiamine HCI 4.00 4.00 4.00 4.00 4.00

* Values shown are in conformance with the Tissue Culture Standards Committee, In Vitro 9:6. 1. Dulbecco, R., Freeman, G. (1959) Virology 8, 396. Smith, J.D., Freeman, G., Vogt, M., and Dulbecco, R. (1960) Virology 12, 185. Tissue Culture Standards Committee, In Vitro 6:2, 93.


56


Advanced D-MEM/F-12Base cat. no. 12634


Inorganic salts

Calcium Chloride (CaCl2) (anhyd.) 116.6

Cupric SULFATE (CuSO4-5H2O) 0.0013

Ferric Nitrate (Fe(NO3)3 • 9H2O 0.05

Ferric sulfate (FeSO4-7H2O) 0.417

Magnesium Chloride (anhydrous) 28.64

Magnesium Sulfate(MgSO4) (anhyd.) 48.84

Potassium Chloride (KCI) 311.8

Sodium Bicarbonate (NaHCO2) 2438

Sodium Chloride (NaCI) 6995.5

Sodium Phosphate dibasic (Na2 HPO4) anhydrous 71.02

Sodium Phosphate monobasic (NaH2 PO4-HO2) 62.5

Zinc sulfate (ZnSO4 7H2O) 0.864

Other components

D-Glucose (Dextrose) 3151

Ethanolamine 1.9

Hypoxanthine Na 2.39

Linoleic Acid 0.042

Lipoic Acid 0.105

Phenol Red 8.1

Putrescine 2HCI 0.081

Sodium Pyruvate 110

Thymidine 0.365

Amino acids

Glycine 18.75

L-Alanine 4.45

L-Arginine hydrochloride 147.5

L-Asparagine-H2O 7.5


L-Cystine hydrochloride-H2O 17.56

L-Cystine 2HCI 31.29

L-Glutamic Acid 7.35

L-Histidine hydrochloride-H2O 31.48

L-Isoleucine 54.47

L-Leucine 59.05

L-Lysine hydrochloride 91.25

L-Methionine 17.24


L-Proline 17.25

L-Serine 26.25

L-Threonine 53.45

L-Tryptophan 9.02

L-Tyrosine disodium salt dihydrate 55.79

L-Valine 52.85

Vitamins

Ascorbic Acid phosphate 2.5

Biotin 0.0035



Folic Acid 2.65

i-Inositol 12.6

Niacinemide 2.02

Pyridoxine hydrochloride 2

Riboflavin 0.219

Thiamine hydrochloride 2.17

Vitamin B12 0.68

Proteins

AlbuMAX® II 400

Human Transferrin (Halo) 7.5

Insulin Recombinant Full Chain 10

Reducing Agents

Glutathione, monosodium 1

Trace Elements

Ammonium Metavanadate 0.0003

Manganous Chloride 0.00005

Sodium Selenite 0.0005

1. Dulbecco, R. And Freeman, G., (1959) Plaque formation by the polyoma virus.Virology 8:3962. Smith, J. D Freeman, G., Vogt, M., et al., The nucleic acid of polyoma virus. Virology 12:1853. Morton, H.J. (1970) Asurvey of commercially available tissue culture media. In Vitro 6(2):894. Ham, R.G., (1965) Clonal growth of mammalian cells in a chemically defined synthetic medium. Proc. Natl. Acad. Sci,. 53:288


edia3



F-12 Nutrient Mixture (Ham)1

Base cat. no. 21700 21765 31765

Component Powder, mg/L

1X liquid, mg/L

1X liquid, mg/L

Inorganic salts

CaCl2 (anhyd.) 33.22 – –

CaCl2 • 2H2O – 44.00 44.00

CuSO4 • 5H2O 0.003 0.0024 0.0024

FeSO4 • 7H2O 0.834 0.83 0.83

KCI 223.60 223.60 223.60

MgCI2 (anhyd.) 57.22 – –

MgCI2 • 6H2O – 122.00 122.00

NaCI 7599.00 7599.00 7599.00

NaHCO3 – 1176.00 1176.00

Na2 HPO4 (anhyd) 142.04 142.00 142.00

ZnSO4 • 7H2O 0.863 0.86 0.86

Other components

D-Glucose 1802.00 1802.00 1802.00

Hypoxanthine – 4.00 4.00

Hypoxanthine (sodium salt)

4.77 – –

Linoleic Acid 0.084 0.084 0.084

DL-68-Thioctic Acid 0.21 0.20 0.20

Phenol Red 1.20 1.20 1.20

Putrescine 2HCI 0.161 0.161 0.161

Sodium Pyruvate 110.00 110.00 110.00

Thymidine 0.73 0.70 0.70

Amino acids

L-Alanine 8.90 8.90 8.90

L-Arginine • HCI 211.00 211.00 211.00

L-Asparagine • H2O 15.01 – –

L-Asparagine (free base)

– 13.00 13.00

L-Aspartic Acid 13.30 13.00 13.00

L-Cystine • HCI – 36.00 36.00

L-Glutamic Acid HCI • H2O

35.12 – –

L-Glutamine Acid 14.70 14.70 14.70

L-Glutamine 146.00 146.00 –

L-Alanyl-L-Glutamine

– – 217.00

Glycine 7.50 7.50 7.50

L-Histidine HCI • H2O 20.96 21.00 21.00

L-Isoleucine 3.94 4.00 4.00

L-Leucine 13.10 13.00 13.00

L-Lysine • HCI 36.50 36.50 36.50

L-Methionine 4.48 4.50 4.50

L-Phenylalanine 4.96 5.00 5.00

L-Proline 34.50 34.50 34.50

L-Serine 10.50 10.50 10.50

L-Threonine 11.90 12.00 12.00

L-Tryptophan 2.04 2.00 2.00

L-Tyrosine – 5.40 5.40

L-Tyrosine (disodium salt)

7.81 – –

L-Valine 11.70 11.70 11.70

Vitamins

Biotin 0.007 0.0073 0.0073

D-Calcium pantothenatec

0.48 0.50 0.50

Choline Chloride 13.96 14.0 14.00

Folic Acid 1.30 1.30 1.30

i-Inositol 18.00 18.00 18.00

Niacinamide* 0.037 0.036 0.036

Pyridoxine HCI 0.062 0.06 0.06

Riboflavin 0.038 0.037 0.037

Thiamine HCI 0.34 0.30 0.30

Vitamin B12 1.36 1.40 1.40

* Values shown are in conformance with the Tissue Culture Standards Committee, In Vitro 9:6.

1. Dulbecco, R., Freeman, G. (1959) Virology 8, 396. Smith, J.D., Freeman, G., Vogt, M. and Dulbecco, R. (1960) Virology 12, 185. Tissue Culture Standards Committee, In Vitro 6:2, 93.


58


Fischer’s Medium for Leukaemic Cells of Mice1

Base cat. no. 21475

Component 1X liquid,mg/L

Inorganic salts

CaCI2 • 2H2O 91.00

KCI 400.00

MgCI2 • 6H2O 100.00

NaCI 8000.00

NaHCO3 1125.00

NaH2PO4 • 2H2O 77.00

Na2HPO4 (anhyd.) 59.00

Other components

D-Glucose 1000.00

Phenol Red 5.00

Amino acids

L-Arginine • HCI 18.60

L-Asparagine 10.00

L-Cystine 20.00

L-Glutamine 204.00

L-Histidine HCI • H2O 60.00

L-Isoleucine 75.00

L-Leucine 30.00

L-Lysine • HCI 50.00

L-Methionine 100.00


L-Serine 15.00

L-Threonine 30.00

L-Tryptophan 10.00

L-Tyrosine 60.00

L-Valine 70.00

Vitamins

Biotin 0.01



Folic Acid 10.00

i-Inositol 1.50

Niacinamide* 0.50

Pyridoxal HCI 0.50

Riboflavin 0.50

Thiamine HCI 1.00

* Values shown are in conformance with the Tissue Culture Standards Committee, In Vitro 9:6. 1. Dulbecco, R., Freeman, G. (1959) Virology 8, 396. Smith, J.D., Freeman, G., Vogt, M. and Dulbecco, R. (1960) Virology 12, 185. Tissue Culture Standards Committee, In Vitro 6:2, 93.

Glasgow MEM 1, 2 (BHK 21)Base cat. no. 21710 22100

Component 1X liquid,mg/L

Powder,mg/L

Inorganic salts

CaCI2 (anhyd.) – 200.00

CaCI2 • 2H2O 265.00 –

Fe(NO3)3 • 9H20 0.10 0.10

KCI 400.00 400.00

MgSO4 (anhyd.) – 97.67

MGSO4 • 7H2O 200.00 –

NaCI 6400.00 6400.00

NaHCO3 2750.00 2750.00

NaH2PO4 • H2O – 124.00

NaH2PO4 • 2H2O 141.00 –

Other components

D-Glucose 4500.00 4500.00

Phenol Red 15.00 15.00

Amino acids

L-Arginine • HCI 42.00 42.00

L-Cystine 24.00 –

L-Cystine • 2HCI – 31.28

L-Glutamine 292.00 292.00

L-Histidine HCI • H20 21.00 21.00


L-Leucine 52.00 52.40

L-Lysine • HCI 73.00 73.10





L-Tyrosine 36.20 –

L-Tyrosine (disodium salt) – 52.09

L-Valine 46.80 46.80

Vitamins

D-Ca pantothenate 2.00 2.00

Choline Chloride 2.00 2.00

Folic Acid 2.00 2.00


Niacinamide* 2.00 2.00

Pyridoxal HCI 2.00 2.00

Riboflavin 0.20 0.20

Thiamine HCI 2.00 2.00

* Values shown are in conformance with the Tissue Culture Standards Committee, In Vitro 9:6.

1. Dulbecco, R., Freeman, G. (1959) Virology 8, 396. Smith, J.D., Freeman, G., Vogt, M. and Dulbecco, R. (1960) Virology 12, 185. Tissue Culture Standards Committee, In Vitro 6:2, 93


edia3



F-10 Nutrient Mixture (Ham)1

Base cat. no. 22390 31550 41550

Component 1X liquid, mg/L 1X liquid, mg/L 1X liquid, mg/LInorganic salts

CaCl2 • 2H2O 44.00 44.00 44.00

CuSO4 • 5H2O 0.0025 0.0025 0.0025

FeSO4 • 7H2O 0.834 0.834 0.834

KCI 285.00 285.00 285.00

KH2PO4 83.00 83.00 83.00

MgSO4 • 7H2O 153.00 153.00 153.00

NaCI 6900.00 7400.00 7400.00

NaHCO3 1200.00 1200.00 1200.00

Na2 HPO4 (anhyd) 154.50 154.50 154.50

ZnSO4 • 7H2O 0.0288 0.0288 0.0288

Other components

D-Glucose 1100.00 1100.00 1100.00

HEPES 5958.00 – –

Hypoxanthine 4.00 4.08 4.00

DL-68-Thioctic Acid 0.20 0.20 0.20

Phenol Red 1.20 1.20 1.20

Sodium Pyruvate 110.00 110.00 110.00

Thymidine 0.73 0.73 0.73

Amino acids

L-Alanine 8.92 9.00 9.00

L-Arginine • HCI 211.00 211.00 211.00

L-Asparagine 12.98 15.01 15.01

L-Aspartic Acid 13.30 13.00 13.00

L-Cystine 25.00 25.00 25.00

L-Glutamic Acid 14.70 14.70 14.70

L-Glutamine 146.00 146.00 –

L-Alanyl-L-Glutamine – – 217.00

L-Glycine 7.52 7.50 7.50

L-Histidine HCI • H2Ob 23.00 23.00 23.00

L-Isoleucine 2.60 2.60 2.60

L-Leucine 13.10 13.00 13.00

L-Lysine • HCI 29.30 29.00 29.00

L-Methionine 4.48 4.50 4.50


L-Proline 11.50 11.50 11.50

L-Serine 10.50 10.50 10.50

L-Threonine 3.58 3.60 3.60


L-Tyrosine 1.81 1.80 1.80

L-Valine 3.50 3.50 3.50

Vitamins

Biotin 0.024 0.024 0.024

D-Calcium pantothenatec

0.72 0.70c 0.70c

Choline Chloride 0.70 0.70 0.70

Folic Acid 1.32 1.32 1.32

i-Inositol 0.54 0.50 0.50

Niacinamide* 0.62 0.60 0.60

Pyridoxine HCI 0.21 0.20 0.20

Riboflavin 0.38 0.40 0.40

Thiamine HCI 1.01 1.00 1.00

Vitamin B12 1.36 1.40 1.40

* Values shown are in conformance with the Tissue Culture Standards Committee, In Vitro 9:6. 1. Dulbecco, R., Freeman, G. (1959) Virology 8, 396. Smith, J.D., Freeman, G., Vogt, M. and Dulbecco, R. (1960) Virology 12, 185. Tissue Culture Standards Committee, In Vitro 6:2, 93.


60


Insect Cell Culture MediaBase cat. no. 11595 11605 11300 11667 11405 21720

Component

Grace’s1

unsupplemented 1X liquid, mg/L

Grace’s 1 supplemented 1X liquid, mg/L

Grace’s 1 powder, mg/L

Grace’s 1 2X liquid, mg/L

IPL-41 1X liquid, mg/L

Schneider’s † ‡ 2

1X liquid, mg/L

Inorganic salts

CaCl2 750.00 500.00 750.00 1000.00 500.00 –

CaCl2 • 2H2O – – – – – 794.00

CoCl2•6H2O – – – – 0.05 –

FeSO4•7H2O – – – – 0.55 –

KCl 4100.00 4100.00 4100.00a 5600.00 1200.00 1600.00

KH2PO4 – – – – – 450.00

MgCl2 – – 1070.00 2140.00 – –

MgCl2 • 6H2O 2280.00 2280.00 – – – –

MgSO4 – – 1360.00 2716.00 918.00 –

MgSO4 • 7H2O 2780.00 2780.00 – – – 3700.00

MnCl2 • 4H2O – – – – 0.02 –

NaCl – – – – 2850.00 2100.00

NaHCO3 350.00 350.00 – 700.00 350.00 400.00

NaH2PO4 • H2O 1013.00 1013.00 1013.00 2026.00 1160.00 –

NaH2PO4 • 2H2O – – – – – –

NaH2PO4 • 7H2O – – – – – 1321.00d

(NH4)6 (Mo7 O24 . 4H2O) – – – – 0.04 –

ZnCl2 – – – – 0.04 –

Other components

Fructose 400.00 400.00 400.00 800.00 – –

Fumaric acid 55.00 55.00 55.00 110.00 4.40 100.00

D-Glucose 700.00 700.00 700.00 1400.00 2500.00 2000.00

α-Ketoglutaric acid 370.00 370.00 370.00 740.00 29.60 200.00

Lactalbumin Hydrolysate – 3330.00 – 6660.00 – –

Malic acid 670.00 670.00 670.00 1340.00 53.60 100.00

Maltose – – – – 1000.00 –

Succinic acid 60.00 60.00 60.00 120.00 4.80 100.00

Sucrose 26680.00 26680.00 26680.00 53360.00 1650.00 –

Trehalose – – – – – 2000.00

Tryptose Broth – – – – – –

Yeastolate – 3330.00 – 6660.00 – –

Amino acids

L-Alanine 225.00 225.00 225.00 450.00 – –

β-Alanine 200.00 200.00 200.00 400.00 300.00 500.00

L-Arginine – – – – – 400.00

L-Arginine • HCl 700.00 700.00 700.00 1400.00 800.00 –

L-Asparagine 350.00 350.00 350.00 700.00 1300.00 –

L-Aspartic acid 350.00 350.00 350.00 700.00 1300.00 400.00

L-Cysteine – – – – – 60.00

L-Cystine 22.00 22.00 – – – 100.00

L-Cystine • 2HCl – – 28.68 57.36 – –

L-Cystine • 2Na – – – – 130.35 –

L-Glutamic acid 600.00 600.00 600.00 1200.00 1500.00 800.00

L-Glutamine 600.00 600.00 600.00 1200.00 1000.00 1800.00

Glycine 650.00 650.00 650.00 1300.00 200.00 250.00

1. Grace, T. CC (1962) Nature 195, 788.a. Increased from 2240.00 mg/L upon recommendation of Doctor Vaughan2. Schneider, Imogene (1964) Exp, Zool, 156:1:9a. Revision of original formulation, Scneider, Imogen (1965) personal communications b. Original formulations list Na2HPO4 at 700.00 mg/L. Revise formulation believed by author to possess better buffering capacity and better nutritive properties.


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Insect Cell Culture MediaBase cat. no. 11595 11605 11300 11667 11405 21720

Component

Grace’s1

unsupplemented 1X liquid, mg/L

Grace’s 1 supplemented 1X liquid, mg/L

Grace’s 1 powder, mg/L

Grace’s 1 2X liquid, mg/L

IPL-41 1X liquid, mg/L

Schneider’s † ‡ 2

1X liquid, mg/L


L-Histidine 2500.00 2500.00 2500.00 5000.00 200.00 400.00

L-Hydroxyproline – – – – 800.00 –

L-Isoleucine 50.00 50.00 50.00 100.00 750.00 150.00

L-Leucine 75.00 75.00 75.00 150.00 250.00 150.00

L-Lysine • HCl 625.00 625.00 625.00 1250.00 700.00 1650.00

L-Methionine – 50.00 50.00 100.00 1,000.00 800.00

L-Phenylalanine 150.00 150.00 150.00 300.00 1,000.00 150.00

L-Proline 350.00 350.00 350.00 700.00 500.00 1700.00

L-Serine 1100.00 550.00 550.00 1100.00 200.00 250.00

L-Threonine 175.00 175.00 175.00 350.00 200.00 350.00

L-Tryptophan 100.00 100.00 100.00 200.00 100.00 100.00

L-Tyrosine 50.00 50.00 – – – 500.00

L-Tyrosine • 2Na – – 62.00 124.00 – –

L-Valine 100.00 100.00 100.00 200.00 500.00 300.00

Vitamins

Para-aminobenzoic acid 0.02 0.02 0.02 0.04 0.32 –

D-Biotin 0.01 0.01 0.01 0.02 0.16 –

D-Ca pantothenate 0.02 0.02 0.02 0.04 0.008 –

Choline chloride 0.20 0.20 0.20 0.40 20.00 –

Folic acid 0.02 0.02 0.02 0.04 0.08 –

i-Inositol 0.02 0.02 0.02 0.04 0.40 –

Niacin 0.02 0.02 0.02 0.04 0.16 –

Pyridoxine HCl 0.02 0.02 0.02 0.04 0.40 –

Riboflavin 0.02 0.02 0.02 0.04 0.08 –

Thiamine HCl 0.02 0.02 0.02 0.04 0.08 –

Vitamin B12 – – – – 0.24 –

1. Grace, T. CC (1962) Nature 195, 788.a. Increased from 2240.00 mg/L upon recommendation of Doctor Vaughan2. Schneider, Imogene (1964) Exp, Zool, 156:1:9a. Revision of original formulation, Scneider, Imogen (1965) personal communications b. Original formulations list Na2HPO4 at 700.00 mg/L. Revise formulation believed by author to possess better buffering capacity and better nutritive properties.


62


Iscove’s Modified Dulbecco’s Media (IMDM)1,2,3Base cat. no. 20156 21980 31980 42200

Component1X liquid,

mg/L1X liquid,

mg/L1X liquid,

mg/LPowder,

mg/L

Inorganic salts

CaCl2 (anhyd.) – – – 165.00

CaCl2 • 2H2O 219.00 219.00 219.00 –

KCl 330.00 330.00 330.00 330.00

KNO3 0.076 0.076 0.076 0.076

MgSO4 • 2H2O 200.00 200.00 200.00 –

MgSO4 (anhyd.) – – – 97.67

NaCl 4505.00 4505.00 4505.00 4500.00

NaHCO3 3024.00 3024.00 3024.00 –

NaH2PO4 • H2Oc – – – 125.00

NaH2PO4 • 2H2O 141.00 141.00 141.00 –

Na2SeO3 • 5H2O 0.017 0.017 0.017 0.017

Other components

D-Glucose 4500.00 4500.00 4500.00 4500.00

Phenol red – 15.00 15.00 15.00

HEPES 5958.00 5958.00 5958.00 5958.00

Sodium pyruvate 110.00 110.00 110.00 110.00

Amino acids

L-Alanine 25.00 25.00 25.00 25.00

L-Asparagine • H2O 28.40 28.40 28.40 –

L- Asparagine (freebase) – – – 25.00

L- Arginine • H2O 84.00 84.00 84.00 84.00

L-Aspartic acid 30.00 30.00 30.00 30.00

L-Cystine • 2HCl – – – 91.24

L-Cystine 70.00 70.00 70.00 –

L-Glutamic acid 75.00 75.00 75.00 75.00

L-Glutamine 584.00 584.00 – 584.00

L-Alanyl-L-glutamine – – 868.00 –

Glycine 30.00 30.00 30.00 30.00

L-Histidine HCl • H2O 42.00 42.00 42.00 42.00

L-Isoleucine 105.00 105.00 105.00 105.00

L-Leucine 105.00 105.00 105.00 105.00

L-Lysine HCl 146.00 146.00 146.00 146.00

L-Methionine 30.00 30.00 30.00 30.00

L-Phenylalanine 66.00 66.00 66.00 66.00

L-Proline 40.00 40.00 40.00 40.00

L-Serine 42.00 42.00 42.00 42.00

L-Threonine 95.00 95.00 95.00 95.00

L-Tryptophan 16.00 16.00 16.00 16.00

L-Tyrosine (disodium salt) 104.20 104.20 104.20 104.20

L-Valine 94.00 94.00 94.00 94.00

Vitamins

Biotin 0.013 0.013 0.013 0.013

D-Ca pantothenate 4.00 4.00 4.00 4.00

Choline chloride 4.00 4.00 4.00 4.00

Folic acid 4.00 4.00 4.00 4.00

i-Inositol 7.20 7.20 7.20 7.20

Niacinamide* 4.00 4.00 4.00 4.00

Pyridoxal HCl 4.00 4.00 4.00 4.00

Riboflavin 0.40 0.40 0.40 0.40

Thiamine HCl 4.00 4.00 4.00 4.00

Vitamin B12 0.013 0.013 0.013 0.013

1. Dubecco, R. And Freeman, G. (1959) Virology 8, 396. Smith, J. D., Freeman, G., Vogt, M. And Dubecco, R. (1960) Virology 12, 165. Tissue Culture Standards Committee, In Vitro 62 93.

2. Iscove, N, N. Melchers, F,. J. Experimental medicine 147, 923.3. Iscove, N, N Personal Communication.b. Values shown are in conformance with the tissue culture standards committee, In

Vitro (1970) 9.6* Also known as Nicotindamide.


edia3



Leibovitz’s L-15 Media 1

Base cat. no. 11415 21083 31415 41300

Component

1X liquid, mg/L

1X liquid, mg/L

1X liquid, mg/L

Powder, mg/L

Inorganic salts

CaCl2 (anhyd. ) – – – 140.00

CaCl2 • 2H2O 185.00 185.00 185.00 –

KCl 400.00 400.00 400.00 400.00

KH2PO4 60.00 60.00 60.00 60.00

MgCl2 (anhyd.) – – – 93.68

MgCl2 • 6H2O 200.00 200.00 200.00 –

MgSO4 (anhyd.) – – – 97.67

NaCl 8000.00 8000.00 8000.00 8000.00

Na2HPO4 190.00 190.00 190.00 190.12

Other components

D(+)-Galactose 900.00 900.00 900.00 900.00

Phenol red 10.00 – 10.00 10.00

Sodium pyruvate 550.00 550.00 550.00 550.00

Amino acids

L-Alanine 225.00 225.00 225.00 225.00

L-Arginine (free base) 500.00 500.00 500.00 500.00

L-Asparagine 250.00 250.00 250.00 250.00

L-Cysteine (free base) 120.00 120.00 120.00 120.00

L-Glutamine 300.00 300.00 – 300.00

Glycine 200.00 200.00 200.00 200.00

L-Histidine (free base) 250.00 250.00 250.00 250.00

L-Isoleucine 250.00 250.00 250.00 250.00

L-Leucine 125.00 125.00 125.00 125.00

L-Lysine (free base) 75.00 75.00 75.00 75.00

L-Methionine 75.00 75.00 75.00 75.00

L-Phenylalanine 125.00 125.00 125.00 125.00

L-Serine 200.00 200.00 200.00 200.00

L-Threonine 300.00 300.00 300.00 300.00

L-Tryptophan 20.00 20.00 20.00 20.00

L-Tyrosine 300.00 300.00 300.00 300.00

L-Valine 100.00 100.00 100.00 100.00

Vitamins

D-Ca pantothenate 1.00 1.00 1.00 1.00

Choline chloride 1.00 1.00 1.00 1.00

Folic acid 1.00 1.00 1.00 1.00

i-Inositol 2.00 2.00 2.00 2.00

Niacinamide* 1.00 1.00 1.00 1.00

Pyridoxal HCl – 1.00 – –

Pyridoxine HCl 1.00 – 1.00 1.00

Flavin Monoucleotide 0.10 0.10 0.10 0.10

Thiamine Monophosphate HCI

1.00 1.00 1.00 1.00

b. Mono hydrate Form listed by the Tissue Culture Standards Committee, In Vitro (1974) 9..6.* Also known as Nicotindamide.

Improved MEM Zinc Option (Richter’s Modification)1

Base cat. no. 10373


Inorganic salts

CaCl2 200.00

FeCl3 • 6H2O 0.54

KCl 400.00

MgCl2 85.75

MgSO4 12.22

NaCl 6800.00

NaHCO3 2200.00

NaH2PO4 • H2O 150.00

ZnSO4 • H2O 0.10

Other components

D-Glucose 2000.00

Gentamicin sulfate 0.05

Putrescine HCl 0.20


Lipoic acid 0.20

Linoleic acid 0.10

Amino acids

L-Arginine • H2O 126.00


L-Cystine 24.00

L-Glutamine 584.00

L-Histidine HCl • H2O 42.00

L-Isoleucine 52.00

L-Leucine 131.00


L-Methionine 15.00


L-Proline 2.00

L-Serine 42.00

L-Threonine 48.00

L-Tryptophan 10.00

L-Tyrosine 36.00

L-Valine 46.00

Vitamins

Biotin 0.10



Folic acid 1.00

i-Inositol 36.00

Niacinamide* 1.00

Pyridoxal HCl 1.00

Riboflavin 0.10

Thiamine • HCl 1.00

Vitamin B12 0.20

1.Richter, a., Sanford, K., and Evans, V., (1972), J. National Cancer inst. 49, 1705.


64


McCoys 5A Media (modified) 1 ,2,3 Base cat. no. 22330 26600 36600


1X liquid, mg/L

1X liquid, mg/L

Inorganic salts

CaCl2 • 2H2O 132.00 132.00 132.00

KCl 400.00 400.00 400.00

MgSO4 • 7H2O 200.00 200.00 200.00

NaCl 5960.00 6460.00 6460.00

NaHCO3 2200.00 2200.00 2200.00

NaH2PO4 • 2H2O 655.00 649.00 649.00

Other components

Bacto-peptone 600.00 600.00 600.00

HEPES 5960.00 – –

D-Glucose 3000.00 3000.00 3000.00

Glutathione (reduced) 0.50 0.50 0.50

Phenol red 10.00 10.00 10.00

Amino acids

L-Alanine 13.40 13.40 13.40

L-Arginine • HCl 42.10 42.10 42.10

L-Asparaginea 45.00 45.00 45.00

L-Aspartic acid 19.90 19.90 19.90

L-Cysteineb 31.50 31.50 31.50

L-Glutamic Acid 22.10 22.10 22.10

L-Glutamine 219.20 219.20 –

L-Alanyl-L-Glutamine – – 326.00

Glycine 7.50 7.50 7.50

L-Histidine HCl • H2O 20.90 20.90 20.90

L-Hydroxyproline 19.70 19.70 19.70

L-Isoleucine 39.30 39.30 39.30

L-Leucine 39.30 39.30 39.30

L-Lysine • HCl 36.50 36.50 36.50

L-Methionine 14.90 14.90 14.90


L-Proline 17.30 17.30 17.30

L-Serine 26.30 26.30 26.30

L-Threonine 17.90 17.90 17.90


L-Tyrosine 18.10 18.10 18.10

L-Valine 17.60 17.60 17.60

Vitamins

Ascorbic acid 0.50 0.50 0.50

Biotin 0.20 0.20 0.20

Choline chloride 5.00 10.00 10.00

D-Ca pantothenate 0.20 0.20 0.20

Folic acid 9.80 10.00 10.00

i-Inositol 36.00 36.00 36.00

Niacin 0.50 0.50 0.50

Niacinamide 0.50 0.50 0.50

Para-aminobenzoic acid 1.00 1.00 1.00

Pyridoxine HCl 0.50 0.50 0.50

Riboflavin 0.20 0.20 0.20

Thiamine HCl 0.20 0.20 0.20

Vitamin B12 2.3075 2.00 2.001. McCoy, T. A., Maxwell, M. And Kruse, P, F. (1995) Proc. Soc. Exper, Biol, Med.,

100. 115.2. Hsu. T. C. And kellog, D, S, Jr (1960) J. Nat. Cancer inst., 25.2213. Iwakata, S. And Grace, J , T., Jr. (1964) N. Y. J. Med., 64:18, 2279a. HCI Form listed by the Tissue Culture Standards

Committee, In Vitro (1974) 9..6.b. Mono hydrate Form listed by the Tissue Culture Standards Committee,

In Vitro (1974) 9:6.* Also known as Nicotindamide.

MCDB 1 31 MediumBase cat. no. 10372


Inorganic salts

CuSO4 • 5H2O 0.0012

MnSO4 • H2O 0.0002

ZnSO4 • H2O 0.0003

NiCl2 • 6H2O 0.0007

FeSO4 • 7H2O 0.283

Na2SiO3 • 9H2O 2.80

CaCl2 • 2H2O 235.00

KCl 298.00

MgSO4 • 7H2O 2464.00

NaCl 6430.00

NaHCO3 1176.00

Na2HPO4 • 7H2O 134.00

H2SeO3 0.0038

(NH4)6Mo7O24 • 4H2O 0.0037

NH4VO3 0.0006

Other components

D-Glucose 1000.00

Adenine 0.135

Thymidine 0.024

Phenol red 12.40


Lipoic acid 0.0021

Putrescine 2HCl 0.002

Amino acids

L-Alanine 2.70



L-Aspartic acid 13.30

L-Cysteine • 2HCl • H2O 35.00

L-Glutamic acid 4.00

Glycine 2.30

L-Histidine HCl•H2O 42.00

L-Isoleucine 66.00

L-Leucine 131.00

L-Lysine•HCl 182.00

L-Methionine 15.00


L-Proline 11.50

L-Serine 32.00

L-Threonine 12.00

L-Tryptophan 4.10

L-Tyrosine 18.10

L-Valine 12.00

Vitamins

Biotin 0.0073



Folinic Acid 0.60

i-Inositol 7.20

Niacinamide* 6.10

Pyridoxine HCl 2.10

Riboflavin 0.0038

Thiamine • HCl 3.40

Vitamin B12 0.0036* Also known as Nicotindamide


edia3



Media 199 1 with Earle's SaltsBase cat. no. 11043 21157 21180 22340 31100 31150 31153 41150

Component1X liquid,

mg/L2X liquid,

mg/L10X liquid,

mg/L1X liquid,

mg/LPowder,

mg/L1X liquid,

mg/L1X liquid,

mg/L1X liquid,

mg/L

Inorganic salts

CaCl2 • H2O – 528.00 2640.00 – – 264.00 264.00 264.00

CaCl2 (anhyd) 200.00 – – 264.00 200.00 – – –

Fe(NO3)3 • 9H2O 0.72 1.40 7.20 0.10 0.72 0.70 0.70 0.70

KCl 400.00 800.00 4000.00 400.00 400.00 400.00 400.00 400.00

KH2PO4 – – – – – – – –

MgSO4 • 7H2O 400.00 – 2000.00 – – 200.00 200.00 200.00

MgSO4 (anhyd.) 98.00 – – 200.00 97.67 – – –

NaCl 6800.00 13600.00 6800.00 6300.00 6.800.00 6800.00 6800.00 6800.00

NaHCO3 2200.00 2500.00 – 2200.00 – 2200.00 1250.00 2200.00

Na2HPO4 • 2H2O – 316.00 1,580.00 158.00 – 158.00 140.00 158.00

NaH2PO4 • H2O 140.00 – – – 140.00 – – –

Other components

Adenine sulfate 10.00 20.00 100.00 10.00 10.00 10.00 10.00 10.00

Adenosine-5-triphosphate 1.00 2.00 10.00 1.00 1.00 1.00 1.00 1.00

Adenosine-5-phosphate 0.20 0.40 2.00 0.20 0.20 0.20 0.20 0.20

Cholesterol 0.20 0.40 2.00 0.20 0.20 0.20 0.20 0.20

Deoxyribose 0.50 1.00 5.00 0.50 0.50 0.50 0.50 0.50

D-Glucose 1000.00 2000.00 10000.00 1000.00 1000.00 1000.00 1000.00 1000.00

Glutathione (reduced) 0.05 0.10 0.50 0.05 0.05 0.05 0.05 0.05

Guanine – 0.60 3.00 – – 0.30 0.30 0.30

Guanine • HCl 0.30 – – 0.30 0.30 – – –

HEPES – – – 5960.00 – – – –

Hypoxanthine – 0.60 3.00 0.30 0.40 – 0.30 0.30

Hypoxanthine • Na 0.40 – – – 0.354 – – –

Phenol red – – 200.00 20.00 20.00 20.00 20.00 20.00

Ribose 0.50 1.00 5.00 0.50 0.50 0.50 0.50 0.50

Sodium Acetate • 3H2O – 166.00 830.00 – 50.00 83.00 83.00 83.00

Sodium Acetate 50.0 – – 83.0 0.30 – 0.30 –

Thymine 0.30 0.60 3.00 0.30 – 0.30 – 0.30

Tween 80® 20.00 40.00 200.00 20.00 20.00 20.00 20.00 20.00

Uracil 0.30 0.60 3.00 0.30 0.30 0.30 0.30 0.30

Xanthine – 0.60 3.00 0.30 – 0.30 0.30 0.30

Xanthine • Na 0.34 – – – 0.344 – – –

Amino acids

L-Alanine 25.00 50.00 250.00 25.00 25.00 25.00 25.00 25.00

L-Arginine • HCl 70.00 140.00 700.00 70.00 70.00 70.00 70.00 70.00

L-Aspartic Acid 30.00 60.00 300.00 30.00 30.00 30.00 30.00 30.00

L-Cysteine HCl • H2O 0.10 – – – 0.11 – – –

L-Cystine • HCl – 22.00 1.10 0.11 – 0.10 0.10 0.10

L-Cystine – 40.00 200.00 20.00 – 20.00 20.00 20.00

L-Cystine 2HCl 26.00 – – – 26.06 – – –

L-Glutamic acid 75.00 119.07 592.43 59.54 66.82 59.24 59.24 59.24

1. Dulbecco, R., Freeman, G. (1959) Virology 8, 396. Smith, J. D., Freeman, G., Vogt, M. And Dulbecco, R. (1960) Virology 12, 185. Tissue Culture Standards Committee, In Vitro 62, 93. a. Value shown are in conformance with the Tissue Culture Standards Committee, In Vitro 96 *Also known as Nicotindamide.


66


Media 199 1 with Earle's SaltsBase cat. no. 11043 11043 11043 11043 11043 11043 31153 41150


2X liquid, mg/L

10X liquid, mg/L

1X liquid, mg/L

Powder, mg/L

1X liquid, mg/L

1X liquid, mg/L

1X liquid, mg/L


L-Glutamine 100.00 200.00 1000.00 100.00 100.00 100.00 100.00 –

Glycine 50.00 100.00 500.00 50.00 50.00 50.00 50.00 50.00

L-Histidine HCl • H2O 22.00 43.76 218.80 21.88 21.88 21.88 21.88 21.88

L-Hydroxyproline 10.00 20.00 100.00 10.00 10.00 10.00 10.00 10.00

L-Isoleucine 40.00 80.00 400.00 40.00 40.00 40.00 40.00 40.00

L-Leucine 60.00 120.00 600.00 60.00 60.00 60.00 60.00 60.00

L-Lysine•HCl 70.00 140.00 700.00 70.00 70.00 70.00 70.00 70.00

L-Methionine 15.00 30.00 150.00 15.00 15.00 15.00 15.00 15.00

L-Phenylalanine 25.00 50.00 250.00 25.00 25.00 25.00 25.00 25.00

L-Proline 40.00 80.00 400.00 40.00 40.00 40.00 40.00 40.00

L-Serine 25.00 50.00 250.00 25.00 25.00 25.00 25.00 25.00

L-Threonine 30.00 60.00 300.00 30.00 30.00 30.00 30.00 30.00

L-Tryptophan 10.00 20.00 100.00 10.00 10.00 10.00 10.00 10.00

L-Tyrosine – 80.00 400.00 40.00 – 40.00 40.00 40.00

L-Tyrosine • 2Na • 2H2O 58.00 – – – 57.88 – – –

L-Valine 25.00 50.00 250.00 25.00 25.00 25.00 25.00 25.00

Vitamins

Ascorbic acid 0.05 0.10 0.50 0.05 0.05 0.05 0.05 0.05

α-Tocopherol phosphate (sodium salt) 0.01 – 0.10 0.01 0.01 0.01 0.01 0.01

Biotin 0.01 0.02 0.10 0.01 0.01 0.01 0.01 0.01

Calciferol 0.10 0.20 1.00 0.10 0.10 0.10 0.10 0.10

D-Ca pantothenate 0.01 0.02 0.10 0.01 0.01 0.01 0.01 0.01

Choline chloride 0.50 1.00 5.00 0.50 0.50 0.50 0.50 0.50

Folic acid 0.01 0.02 0.10 0.01 0.01 0.01 0.01 0.01

i-Inositol 0.05 0.10 0.50 0.05 0.05 0.05 0.05 0.05

Menadione 0.01 0.02 0.10 0.01 0.01 0.01 0.01 0.01

Niacin 0.025 0.05 0.25 0.025 0.025 0.025 0.025 0.025

Niacinamide* 0.025 0.025 0.30 0.025 0.025 0.025 0.025 0.025

Para-aminobenzoic Acid 0.05 0.10 0.50 0.05 0.05 0.05 0.05 0.05

Pyridoxal HCl 0.025 0.05 0.30 0.025 0.025 0.025 0.025 0.025

Pyridoxine HCl 0.025 0.05 0.30 0.025 0.025 0.025 0.025 0.025

Riboflavin 0.01 0.02 0.10 0.01 0.01 0.01 0.01 0.01

Thiamine HCl 0.01 0.02 0.10 0.01 0.01 0.01 0.01 0.01

Vitamin A (acetate)a 0.14 0.20 1.00 0.10 0.14 0.10 0.10 0.10

1. Dulbecco, R., Freeman, G. (1959) Virology 8, 396. Smith, J. D., Freeman, G., Vogt, M. And Dulbecco, R. (1960) Virology 12, 185. Tissue Culture Standards Committee, In Vitro 62, 93.

a. Value shown are in conformance with the Tissue Culture Standards Committee, In Vitro 96* Also known as Nicotindamide.


edia3



Minimum Essential Medium (MEM) α Media 1

Base cat. no. 11900 22561 22571 32561 32571 41061


1X liquid, mg/L

1X liquid, mg/L

1X liquid, mg/L

1X liquid, mg/L

1X liquid, mg/L

Inorganic salts

CaCl2 (anhyd.) 200.00 – – – – –

CaCl2 • 2H2O – 264.00 264.00 264.00 264.00 264.00

KCl 400.00 400.00 400.00 400.00 400.00 400.00

MgSO4 (anhyd.) 97.67 – – – – –

MgSO4 • 7H2O – 200.00 200.00 200.00 200.00 200.00

NaCl 6800.00 6800.00 6800.00 6800.00 6800.00 6800.00

NaHCO3– 2200.00 2200.00 2200.00 2200.00 2200.00

Na2HPO4 • H2O 140.00 – – – – –

Na2HPO4 • 2H2O – 158.00 158.00 158.00 158.00 158.00

Other components

D-Glucose 1000.00 1000.00 1000.00 1000.00 1000.00 1000.00

DL-68 Thioctic Acid 0.20 0.20 0.20 0.20 0.20 0.20

Phenol red 10.00 10.00 10.00 10.00 10.00 –

Sodium Pyruvate 110.00 110.00 110.00 110.00 110.00 110.00

Amino acids

L-Alanine 25.00 25.00 25.00 25.00 25.00 25.00

L-Arginine • HCl 126.98 127.00 127.00 127.00 127.00 127.00

L-Asparagine • H2O 50.00 25.00 50.00 25.00 50.00 50.00

L-Aspartic acid 30.00 30.00 30.00 30.00 30.00 30.00

L-Cystine – 24.00 24.00 24.00 24.00 –

L-Cystine • 2HCl 31.00 – – – – 31.00

L-Cysteine HCl – – – 100.00 100.00 –

L-Cysteine HCl • H2O 100.00 100.00 100.00 – – 100.00

L-Glutamic acid 75.00 75.00 75.00 75.00 75.00 75.00

L-Glutamine 292.00 292.00 292.00 – – 292.00

L-Alanyl-L-glutamine – – – 434.00 434.00 –

Glycine 50.00 50.00 50.00 50.00 50.00 50.00

L-Histidine HCl • H2O 41.88 42.00 42.00 42.00 42.00 42.00

L-Isoleucine 52.40 53.00 53.00 53.00 53.00 53.00

L-Leucine 52.40 52.00 52.00 52.00 52.00 52.00

L-Lysine•HCl 72.47 73.00 73.00 73.00 73.00 73.00

L-Methionine 15.00 15.00 15.00 15.00 15.00 15.00

L-Phenylalanine 32.00 32.00 32.00 32.00 32.00 32.00

L-Proline 40.00 40.00 40.00 40.00 40.00 40.00

L-Serine 25.00 25.00 25.00 25.00 25.00 25.00

L-Threonine 48.00 48.00 48.00 48.00 48.00 48.00

L-Tryptophan 10.00 10.00 10.00 10.00 10.00 10.00

L-Tyrosine – 36.00 36.00 36.00 36.00 36.00

L-Tyrosine (disodium salt)

52.09 – – – – –

L-Valine 46.00 46.00 46.00 46.00 46.00 46.00

1. Nature, new biology (1971) 230, 52.


68


Base cat. no. 11900 22561 22571 32561 32571 41061


1X liquid, mg/L

1X liquid, mg/L

1X liquid, mg/L

1X liquid, mg/L

1X liquid, mg/L

Vitamins

L-Ascorbic acid 50.00 50.00 50.00 50.00 50.00 50.00

Biotin 0.10 0.10 0.10 0.10 0.10 0.10

D-Ca pantothenate 1.00 1.00 1.00 1.00 1.00 1.00

Choline chloride 1.00 1.00 1.00 1.00 1.00 1.00

Folic acid 1.00 1.00 1.00 1.00 1.00 1.00

i-Inositol 2.00 2.00 2.00 2.00 2.00 2.00

Niacinamide* 1.00 1.00 1.00 1.00 1.00 1.00

Pyridoxal HCl 1.00 1.00 1.00 1.00 1.00 1.00

Riboflavin 0.10 0.10 0.10 0.10 0.10 0.10

Thiamine HCl 1.00 1.00 1.00 1.00 1.00 1.00

Vitamin B121.36 1.40 1.40 1.40 1.40 1.40

Ribonucleosides

Adenosine 10.00 – 10.00 – 10.00 10.00

Cytidine 10.00 – 10.00 – 10.00 10.00

Guanosine 10.00 – 10.00 – 10.00 10.00

Uridine 10.00 – 10.00 – 10.00 10.00

Deoxyribonucleosides

2´ Deoxyadenosine 10.00 – 10.00 – 10.00 10.00

2´ Deoxycytidine HCl 11.00 – 11.00 – 11.00 11.00

2´ Deoxyguanosine 10.00 – 10.00 – 10.00 10.00

2´ Deoxythymidine 10.00 – 10.00 – 10.00 10.00

1. Nature, new biology (1971) 230, 52.


edia3



Medium 1991 with Hanks' SaltsBase cat. no. 10012 22350


1X Liquid, mg/L

Inorganic salts

CaCl2 • H2O – 185.00

CaCl2 (anhyd.) 140.10 –

Fe(NO3)3 • 9H2O 0.72 0.10

KCl 400.00 400.00

KH2PO460.00 60.00

MgSO4 • 7H2O 97.70 200.00

MgSO4 (anhyd.) – –

NaCl 7500.00 7500.00

NaHCO3– 350.00

Na2HPO4 • 2H2O – –

Na2HPO4 • H2O – –

Na2HPO4 (anhyd.) 47.40 48.00

Other components

Adenine Sulphate 10.00 10.00

Adenosine-5-triphosphate 1.00 1.00

Adenosine-5-phosphate 0.20 0.20

Cholesterol 0.20 0.20

Dexoxyriboxe 0.50 0.50

D-Glucose 1000.00 1000.00

Glutathione (reduced) 0.05 0.05

Guanine – –

Guanine • HCI 0.30 0.30

HEPES 5958.00 5960.00

Hypoxanthine – 0.30

Hypoxanthine • Na 0.344 –


Ribose 50.0 50.0

Sodium Acetate • 3H2O – –

Sodium Acetate 50.00 83.00

Thymine 30.0 30.0

Tween 80® 20.0 200.00

Uracil 30.0 30.0

Xanthine – 30.0

Xanthine•Na 0.344 –

Amino acids

L-Alanine 25.00 25.00

L-Arginine • HCl 70.00 70.00

L-Aspartic acid 30.00 30.00

L-Cysteine HCl • H2O 0.11 0.11

L-Cysteine HCl – –

L-Cystine – 20.00

L-Cystine • 2HCl 26.06 –

L-Glutamic acid 66.82 59.54

L-Glutamine 100.00 100.00

L-Alanyl-L-glutamine – –

Glycine 50.00 50.00

L-Histidine HCl • H2O 21.88 21.88

L-Hydroxyproline 10.00 10.00


L-Leucine 60.00 60.00

L-Lysine • HCl 70.00 70.00



L-Proline 40.00 40.00

L-Serine 25.00 25.00


L-Thyptophan 10.00 10.00

L-Thyrosine – 40.00

L-Thyrosine • Na • 2H2O 57.88 –

L-Valine 25.00 25.00

Vitamins

Ascorbic Acid 0.05 0.05

α- Tocopherol Phosphate (disodium salt)

0.01 0.01

Biotin 0.01 0.01

Calciferol 10.00 10.0

D-Ca Pantothenate 0.01 0.01




Menadione 0.01 0.01

Niacin 0.025 0.025


Para-aminobenzoic Acid 0.05 0.05

Pyridoxal HCI 0.025 0.025

Pyridoxine HCI 0.025 0.025


Thiamine HCI 0.01 0.01

Vitamin A (acetate)a 0.14 0.10

1. Dulbecco, R., Freeman, G. (1959) Virology 8, 396. Smith, J. D., Freeman, G., Vogt, M. And Dulbecco, R. (1960) Virology 12, 185. Tissue Culture Standards Committee, In Vitro 62, 93.

a. Value shown are in conformance with the Tissue Culture Standards Committee, In Vitro 96* Also known as Nicotindamide.


70


Modified Eagle Medium (MEM), 2X 1 with Earle’s Modified SaltsBase cat. no. 21935


Inorganic salts

CaCl2 • 2H2O 376.00

Fe(NO3)3 • 9H2O 1.00

KCl 800.00

MgSO4•7H2O 400.00

NaCl 12800.00

NaHCO3 8400.00

NaH2PO4 • H2O 238.00

Other components

D-Glucose 9000.00

Amino acids

L-Arginine 420.00

L-Cystine 96.00

L-Glutamine 870.00

L-Histidine 124.00

L-Isoleucine 210.00

L-Leucine 209.60

L-Lysine 232.00

L-Methionine 60.00


L-Threonine 192.00

L-Tryptophan 40.00

L-Tyrosine 144.00

L-Valine 184.00

Vitamins



Folic acid 4.00

i-Inositol 8.00

Niacinamide* 4.00

Pyridoxal HCl 4.00

Riboflavin 0.40

Thiamine HCl 4.00

1. Daniel, M. D. And Melendez V. V. (1969) Proc, Soc, Exp, Biol, Med. 127:33.

* Also known as Nicotindamide.

Base cat. no. 21340


Inorganic salts

CaCl2 200.00

KCl 400.00

MgSO4 97.67

NaCl 6800.00

NaHCO3 2200.00

NaH2PO4 • H2O 140.00

Other components

D-Glucose 1000.00

D-Glucuronolactone 1.80

Phenol red (sodium salt) 20.00

Sodium acetate 50.00

Sodium glucuronate • H2O 1.80

Tween 80® 12.50

Coenzymes

CoA (coenzyme A) 2.50

DPN (diphosphopyridine nucleotide) 7.00

FAD (flavin adenine dinucleotide) 1.00

TPN (triphosphopyridine nucleotide monosodium)

1.00

TPP (cocarboxylase) 1.00

UTP (uridine triphosphate, sodium) 1.00

Reducing agents

Ascorbic acid 50.00

Glutathione, monosodium 10.00

Nucleic acid derivatives

Deoxyadenosine 10.00

Deoxyguanosine 10.00

Deoxycytidine HCl 10.00

5-Methylcytosine 0.10

Thymidine 10.00

Amino acids

L-Alanine 31.60

L-α-Amino-n-butyric acid 5.50

L-Arginine HCl 31.20

L-Asparagine•H2O 9.20


L-Cystine•2HCl 14.00

L-Cystine•HCI•H2O 290.00

D-Glucosamine•HCl 3.90

Base cat. no. 21340




Glycine 13.60

L-Histidine • HCl • H2O 26.70

Hydroxy-L-Proline 4.10

L-Isoleucine 18.00

L-Leucine 20.40


L-Methionine 4.50

L-Ornithine • HCl 9.40


L-Proline 6.20

L-Serine 10.80

L-Taurine 4.20

L-Threonine 18.90

L-Tryptophan 17.50

L-Tyrosine • 2Na • 2H2O 21.00

L-Valine 25.00

Vitamins

α-Tocopherol phosphate (disodium salt)

0.025

d-Biotin 0.025

Calciferol (Vitamin D2) 0.250



Folic acid 0.025

i-Inositol 0.125

Menadione (Vitamin K) 0.025

Niacin 0.063

Niacinamide* 0.063

Para-aminobenzoic acid 0.125

Pyridoxal HCl 0.063

Pyridoxine HCl 0.063

Riboflavin 0.025

Thiamine HCl 0.025

Vitamin A (crystallized alcohol) 0.25

Vitamin B12 10.00

* Also known as Nicotindamide.

Tween 80® is a registered trademark of ICI Americas

NCTC-109 Medium


edia3



Minimum Essential Media1 (MEM)Base cat. no. 10370 11012 11380 11700 21011 21090 21430 21575


Powder, mg/L

1X liquid, mg/L

Powder, mg/L

Powder, mg/L

1X liquid, mg/L

10X liquid, mg/L

1X liquid, mg/L

Inorganic salts

CaCl2 • 2H2O – – – – – 264.00 2640.00 185.00

CaCl2 (anhyd.) 200.00 140.00 – 200.00 200.00 200.00 – –

KCl 400.00 400.00 400.00 400.00 400.00 400.00 400.00 400.00

KH2PO4 – 60.00 – – – – – 60.00

MgSO4 (anhyd.) 98.00 97.67 98.00 97.67 97.67 – – –

MgSO4 • 7H2O – – – – – 200.00 2,000.00 100.00

MgCI2 • 6H2O – – – – – – – 100.00

NaCl 6800.00 7500.00 6800.00 6800.00 6300.00 6800.00 6,800.00 8000.00

NaHCO3 2200.00 – 2200.00 – – 2200.00 – 350.00

NaH2PO4 • 2H2O – – – – – 158.00 1580.00 –

NaH2PO4 • H2Oa 140.00 – 1400.00 140.00 140.00 – – –

NaH2PO4 – 47.88 – – – – – 48.00

Other components

D-Glucose 1000.00 1000.00 1000.00 1000.00 1000.00 1000.00 1000.00 1000.00

Phenol red 10.00 10.00 10.00 6.00 10.00 10.00 10.00 10.00

Sodium succinate – – – 100.00 – – – –

Succinic acid – – – 75.00 – – – –

HEPES – 5958.00 – – 5958.00 – – –

Amino acids

L-Alanine 9.00 – – – – – – –

L-Arginine • HCl 126.00 126.00 126.00 126.00 126.00 126.00 126.00 126.00

L-Asparagine • H2O 13.00 – – – – – – –

L-Aspartic acid 13.00 – – – – – – –

L-Cystine – – – – – 24.00 24.00 24.00

L-Cystine•2HCl 31.00 31.29 31.00 31.00 31.29 – – –

L-Glutamic acid 15.00 – – – – – – –

L-Glutamine – 292.00 – – 292.00 – – 292.00

Glycine 7.50 – – – – – – –

L-Histidine HCl • H2O 42.00 42.00 42.00 42.00 42.00 42.00 42.00 42.00

L-Isoleucine 52.00 52.00 52.00 52.00 52.00 52.00 52.00 52.00

L-Leucine 52.00 52.00 52.00 52.00 52.00 52.00 52.00 52.00

L-Lysine • HCl 72.50 72.50 73.00 72.50 72.50 73.00 73.00 73.00

L-Methionine 15.00 15.00 15.00 15.00 15.00 15.00 15.00 15.00

L-Phenylalanine 32.00 32.00 32.00 32.00 32.00 32.00 320.00 32.00

L-Proline 12.00 – – – – – – –

L-Serine 11.00 – – – – – – –

L-Threonine 48.00 48.00 48.00 48.00 48.00 48.00 480.00 48.00

L-Tryptophan 10.00 10.00 10.00 10.00 10.00 10.00 100.00 10.00

L-Tyrosine – – – 36.00 – 36.00 360.00 36.00

L-Tyrosine (disodium salt) 52.00 52.09 52.00 52.09 52.00 – – –

L-Valine 46.00 46.00 46.00 46.00 46.00 46.00 46.00 46.00

Vitamins

D-Ca pantothenate 1.00 1.00 1.00 1.00 1.00 1.00 10.00 1.00

Choline bitartrate – – – 1.80 – – – –

Choline chloride 1.00 1.00 1.00 – 1.00 1.00 10.00 1.00

Folic acid 1.00 1.00 1.00 1.00 1.00 1.00 10.00 1.00

i-Inositol 2.00 2.00 2.00 2.00 2.00 2.00 20.00 2.00

Niacinamide* 1.00 1.00 1.00 1.00 1.00 1.00 10.00 1.00

1.Eagle, H. (1959) Science, 130, 432.a. Original Formula lists this compnent as NaH2PO4, 2H2O2 * Also known as Nicotindamide.


72


Vitamins, continued

Pyridoxal HCl 1.00 1.00 1.00 1.00 1.00 1.00 10.00 1.00

Riboflavin 0.10 0.10 0.10 0.10 0.10 0.10 1.00 0.10

Thiamine HCl 1.00 1.00 1.00 1.00 1.00 1.00 10.00 1.00

1.Eagle, H. (1959) Science, 130, 432.a. Original Formula lists this compnent as NaH2PO4, 2H2O2 * Also known as Nicotindamide.


edia3



Minimum Essential Media1 (MEM)Base cat. no. 31095 32360 41090 41500 42360 51200 61100


1X liquid, mg/L

1X liquid, mg/L

Powder, mg/L

1X liquid, mg/L

1X liquid, mg/L

Powder, mg/L

Inorganic salts

CaCl2•2H2O 264.00 264.00 264.00 264.00 264.00 264.00 –

CaCl2 (anhyd.) – – – – – – 200.00

KCl 400.00 400.00 400.00 400.00 400.00 400.00 400.00

MgSO4 (anhyd.) – 97.67 – 97.67

MgSO4•7H2O 200.00 200.00 200.00 – 200.00 200.00 –

MgCI2•6H2O – – – – – – –

NaCl 6,800.00 6,300.00 6,800.00 6,800.00 6,300.00 6,800.00 6,800.00

NaHCO32,200.00 2,200.00 2,200.00 – 2,200.00 2,200.00 –

NaH2PO4•2H2O 158.00 158.00 158.00 140.00 158.00 158.00 140.00

NaH2HPO4•2H2Oa – – – – – – –

Other components

D-Glucose 1,000.00 1,000.00 1,000.00 1,000.00 1,000.00 1,000.00 1,000.00

HEPES – 5,960.00 – – 5,960.00 – –

Phenol red 10.00 10.00 10.00 10.00 10.00 – 10.00

Amino acids

L-Alanine – – – 8.90 – – –

L-Arginine • HCl 126.40 126.40 126.00 126.40 126.40 126.40 126.00

L-Asparagine • H2O – – – 13.02 – – –

L-Aspartic acid – – – 13.30 – – –

L-Cystine 24.02 24.02 24.02 – 24.02 – –

L-Cystine•2HCl – – – 31.29 – 24.02 31.29

L-Glutamic acid – – – 14.70 – – –

L-Glutamine 292.00 – – 292.00 – – 292.00

L-Alanyl-L-glutamine – – 406.00 – – – –

Glycine – – – 7.50 434.00 – –

L-Histidine HCl•H2O 41.92 41.92 41.92 42.00 41.92 41.92 42.00

L-Isoleucine 52.46 52.46 52.46 52.00 52.46 52.46 52.00

L-Leucine 52.46 52.46 52.46 52.00 52.46 52.46 52.00

L-Lysine•HCl 73.06 73.06 73.06 72.50 73.06 73.06 72.50

L-Methionine 14.92 14.92 14.92 15.00 14.92 14.92 15.00

L-Phenylalanine 33.02 33.02 32.02 32.00 33.02 33.02 32.00

L-Proline – – – 11.50 – – –

L-Serine – – – 10.50 – – –

L-Threonine 47.64 47.64 47.64 48.00 47.64 47.64 48.00

L-Tryptophan 10.20 10.20 10.20 10.00 10.20 10.20 10.00

L-Tyrosine 36.22 36.22 36.22 – 36.22 36.22 –

L-Tyrosine (disodium salt) – – – 52.09 – – 51.09

L-Valine 46.86 46.86 46.86 46.00 46.86 46.86 46.00

Vitamins

D-Ca pantothenate 1.00 1.00 1.00 1.00 1.00 1.00 1.00

Choline chloride 1.00 1.00 1.00 1.00 1.00 1.00 1.00

Folic acid 1.00 1.00 1.00 1.00 1.00 1.00 1.00

i-Inositol 2.00 2.00 2.00 2.00 2.00 2.00 2.00

Niacinamide* 1.00 1.00 1.00 1.00 1.00 1.00 1.00

Pyridoxal HCl 1.00 1.00 1.00 1.00 1.00 1.00 1.00

Riboflavin 0.10 0.10 0.10 0.10 0.10 0.10 0.10

Thiamine HCl 1.00 1.00 1.00 1.00 1.00 1.00 1.00

1.Eagle, H. (1959) Science, 130, 432.a. Original Formula lists this component as NaH2PO4, 2H2O2 * Also known as Nicotindamide.


74


Base cat. no. 41350


Inorganic salts

CaCl2 • 2H2O 264.00

Fe(NO3)3• 9H2O 0.10

KCl 400.00

MgSO4 • 7H2O 200.00

NaCl 6800.00

NaHCO3 2200.00

NaH2PO4 • 2H2O 158.00

Other components

Ethyl Alcohol 40.00

D-Glucose 1000.00

D-Glucuronolactone 1.80

Phenol Red (sodium salt) 20.00

Sodium Acetate • 3H2O 50.00

Sodium Glucuronate • H2O 1.80

Tween 80® 12.50

Coenzymes

CoA (coenzyme A) 2.50

DPN (diphosphopyridine nucleotide)

7.00

FAD (flavin adenine dinucle-otide)

100

TPN (triphosphopyridine nucleotide monosodium)

1.00

TPP (cocarboxylase) 1.00

UTP (uridine triphosphate, sodium)

1.00

Reducing Agents

Ascorbic Acid 50.00

Glutathione, monosodium 10.00

Nucleic Acid Derivatives

Deoxyadenosine 10.00

Deoxyguanosine 10.00

Deoxycytidine HCI 10.00

5-Methylcytosine 0.10

Thymidine 10.00

Amino Acids

L-Alanine 31.48

L-α-Amino-n-butyric Acid 5.50

L-Arginine HCI 31.20

L-Asparagine 8.10


L-Cystine 10.50


D-Glucosamine • HCI 3.90

L-Glutamic Acid 8.30

L-Glutamine 135.70

Glycine 13.50

L-Histadine • HCI • H20 26.50

NCTC - 1351-4 Medium

Base cat. no. 41350


Hydoxy-L-Proline 4.10

L-Isoleucine 18.00

L-Leucine 20.40

L-Lysine•HCI 38.40

L-Methionine 4.40

L-Ornithine • HCI 9.40


L-Proline 6.10

L-Serine 10.80

L-Taurine 4.20

L-Threonine 18.90

L-Tryptophan 17.50

L-Tryosine 16.40

L-Valine 25.00

Vitamins

α-Tocopherol Phosphate (disodium salt)

0.025

d-Biotin 0.025

Calciferol (Vitamin D2) 0.25

D-Ca-Panthothenate 0.025


Folic Acid 0.025

i-Inositol 0.125

Menadione (Vitamin K) 0.25

Niacin 0.625

Niacinamide* 0.003

Para-aminobenzoic Acid 0.125

Pyridoxal HCI 0.0625

Pyridoxine HCI 0.0625

Riboflavin 0.025

Thiamine HCI 0.025

Vitamin A (crystallised alcohol)

0.25

Vitamin B12 10.00

1. Evans, V.J. et al, (1956) Can. Res., 16,77. 2. Mcquilken, W. T. Et al (1957) J. Nat. Cancer Inst., 19,885.3. Evans V.J ., et al, (1964) Exp. Cell res., 36, 439.4. Tissue Culture Standards Committee, In Vitro (1970)

6.2, 93* Also known as Nicotindamide.


edia3



RPMI 1640 Medium1

Base cat. no. 11835 11879 13018 21875 22409 27016 31870


1X liquid, mg/L

Powder, mg/L

1X liquid, mg/L

1X liquid, mg/L

1X liquid, mg/L

1X liquid, mg/L

Inorganic salts

Ca(NO3)2 • 4H2O 100.00 100.00 100.00 100.00 100.00 100.00 100.00

KCl 400.00 400.00 400.00 400.00 400.00 400.00 400.00

MgSO4 (anhyd.) 48.84 48.84 48.84 – – – –

MgSO4 • 7H2O – – – 100.00 100.00 100.00 100.00

NaCl 6000.00 6000.00 5500.00 6000.00 6400.00 6000.00 6000.00

NaHCO3 2000.00 2000.00 – 2000.00 1000.00 2000.00 2000.00

Na2HPO4 (anhyd.) 800.00 1,512.00 800.00 800.00 800.00 800.00 800.00

Other components

D-Glucose 2000.00 – 2000.00 2000.00 2000.00 2000.00 2000.00

Glutathione (reuduced) 1.00 1.00 1.00 1.00 1.00 1.00 1.00

HEPES – – 5958.00 – 4770.00 – –

Phenol red – 5.00 5.00 5.00 5.00 5.00 5.00

Amino acids

L-Alanine HCI 241.86 200.00 241.86 240.00 240.00 241.86 240.00

L-Asparagine (free base) 50.00 50.00 50.00 50.00 50.00 50.00 50.00

L-Aspartic acid 20.00 20.00 20.00 20.00 20.00 20.00 20.00

L-Cystine • 2HCl 65.15 65.00 65.15 – – – –

L-Glutamic acid 20.00 20.00 20.00 20.00 20.00 20.00 20.00

L-Glutamine 300.00 300.00 300.00 300.00 – 300.00 –

Glycine 10.00 10.00 10.00 10.00 10.00 10.00 10.00

L-Histidine (free base) 15.00 15.00 15.00 15.00 15.00 15.00 15.00

L-Hydroxyproline 20.00 20.00 20.00 20.00 20.00 20.00 20.00

L-Isoleucine 50.00 50.00 50.00 50.00 50.00 50.00 50.00

L-Leucine 50.00 50.00 50.00 50.00 50.00 50.00 50.00

L-Lysine • HCl 40.00 40.00 40.00 40.00 40.00 40.00 40.00

L-Methionine 15.00 15.00 15.00 15.00 15.00 15.00 15.00

L-Phenylalanine 15.00 15.00 15.00 15.00 15.00 15.00 15.00

L-Proline 20.00 20.00 20.00 20.00 20.00 20.00 20.00

L-Serine 30.00 30.00 30.00 30.00 30.00 30.00 30.00

L-Threonine 20.00 20.00 20.00 20.00 20.00 20.00 20.00

L-Tryptophan 5.00 5.00 5.00 5.00 5.00 5.00 5.00

L-Tyrosine – – – 20.00 20.00 20.00 20.00

L-Tyrosine (disodium salt) 28.94 28.00 28.94 – – – –

L-Valine 20.00 20.00 20.00 20.00 20.00 20.00 20.00

Vitamins

D-Biotin 0.20 0.20 0.20 0.20 0.20 0.20 0.20



Folic acid 1.00 1.00 1.00 1.00 1.00 – 1.00

i-Inositol 35.00 35.00 35.00 35.00 35.00 35.00 35.00

Niacinamide* 1.00 1.00 1.00 1.00 1.00 1.00 1.00

Para-aminobenzoic acid 1.00 1.00 1.00 1.00 1.00 1.00 1.00

Pyridoxine HCl 1.00 1.00 1.00 1.00 1.00 1.00 1.00

Riboflavin 0.20 0.20 0.20 0.20 0.20 0.20 0.20

Thiamine HCl 1.00 1.00 1.00 1.00 1.00 1.00 1.00

Vitamin B12 0.005 0.005 0.005 0.005 0.005 0.005 0.005

1. Moore, G. E., .Gerner, R. E. And Franklin, H,. A. (1967) A. M. A., 199 519.* Also known as Nicotindamide.


76


RPMI 1640 Medium1

Base cat. no. 30244 42401 42402 51800 52400 61870 72400


1X liquid, mg/L

1X liquid, mg/L

Powder, mg/L

1X liquid, mg/L

1X liquid, mg/L

1X liquid, mg/L

Inorganic salts

Ca(NO3)2 • 4H2O 100.00 100.00 100.00 100.00 100.00 100.00 100.00

KCl 400.00 400.00 400.00 400.00 400.00 400.00 400.00

MgSO4 (anhyd.) – – – 48.84 – – –

MgSO4 • 7H2O 100.00 100.00 100.00 – 100.00 100.00 100.00

NaCl 6000.00 5500.00 6000.00 6000.00 5500.00 6000.00 5500.00

NaHCO3 2000.00 2000.00 850.00 – 2000.00 2000.00 2000.00

Na2HPO4 (anhyd.) 800.00 800.00 800.00 800.00 800.00 800.00 800.00

Other components

D-Glucose 2000.00 2000.00 2000.00 2000.00 2000.00 2000.00 2000.00

Glutathione (reuduced) 1.00 1.00 1.00 1.00 1.00 1.00 1.00

HEPES – 5958.00 4770.00 – 5958.00 – 5958.00

Phenol red – 5.00 5.00 5.00 5.00 5.00 5.00

Amino acids

L-Arginine HCI 240.00 240.00 240.00 240.00 240.00 240.00 240.00

L-Asparagine (free base) 50.00 50.00 50.00 50.00 50.00 50.00 50.00

L-Aspartic acid 20.00 20.00 20.00 20.00 20.00 20.00 20.00

L-Cystine 50.00 50.00 50.00 – 50.00 50.00 50.00

L-Cystine • 2HCl – – – 65.15 – – –

L-Glutamic acid 20.00 20.00 20.00 20.00 20.00 20.00 20.00

L-Alanyl-L-Glutamine – – – – – 446.00 446.00

L-Glutamine – – – 300.00 300.00 – –

Glycine 10.00 10.00 10.00 10.00 10.00 10.00 10.00

L-Histidine (free base) 15.00 15.00 15.00 15.00 15.00 15.00 15.00

L-Hydroxyproline 20.00 20.00 20.00 20.00 20.00 20.00 20.00

L-Isoleucine 50.00 50.00 50.00 50.00 50.00 50.00 50.00

L-Leucine 50.00 50.00 50.00 50.00 50.00 50.00 50.00

L-Lysine • HCl 40.00 40.00 40.00 40.00 40.00 40.00 40.00

L-Methionine 15.00 15.00 15.00 15.00 15.00 15.00 15.00

L-Phenylalanine 15.00 15.00 15.00 15.00 15.00 15.00 15.00

L-Proline 20.00 20.00 20.00 20.00 20.00 20.00 20.00

L-Serine 30.00 30.00 30.00 30.00 30.00 30.00 30.00

L-Threonine 20.00 20.00 20.00 20.00 20.00 20.00 20.00

L-Tryptophan 5.00 5.00 5.00 5.00 5.00 5.00 5.00

L-Tyrosine 20.00 20.00 20.00 – 20.00 20.00 20.00

L-Tyrosine (disodium salt) – – – 28.94 – – –

L-Valine 20.00 20.00 20.00 20.00 20.00 20.00 20.00

Vitamins

D-Biotin 0.20 0.20 0.20 0.20 0.20 0.20 0.20



Folic acid 1.00 1.00 1.00 1.00 1.00 1.00 1.00

i-Inositol 35.00 35.00 35.00 35.00 35.00 35.00 35.00

Niacinamide* 1.00 1.00 1.00 1.00 1.00 1.00 1.00

Para-aminobenzoic acid 1.00 1.00 1.00 1.00 1.00 1.00 1.00

Pyridoxine HCl 1.00 1.00 1.00 1.00 1.00 1.00 1.00


edia3



RPMI 1640 Medium1

Base cat. no. 30244 42401 42402 51800 52400 61870 72400

Component 1Xliquid, mg/L

1X liquid, mg/L

1X liquid, mg/L

Powder, mg/L

1X liquid, mg/L

1X liquid, mg/L

1X liquid, mg/L

Vitamins, continued

Riboflavin 0.20 0.20 0.20 0.20 0.20 0.20 0.20

Thiamine HCl 1.00 1.00 1.00 1.00 1.00 1.00 1.00

Vitamin B12 0.005 0.005 0.005 0.005 0.005 0.005 0.005

1. Moore, G. E., .Gerner, R. E. And Franklin, H,. A. (1967) A. M. A., 199 519.* Also known as Nicotindamide.


78


Advanced RPMI Medium 1640Base cat. no. 12633


Inorganic salts

Calcium nitrate (Ca(NO3)2

4H2O)100

Magnesium Sulfate (MgSO4-7H2O)

100

Potassium Chloride (KCI) 400

Sodium Bicarbonate (NaHCO3)

2000

Sodium Chloride (NaCI) 6000

Sodium Phosphate dibasic (Na2HPO4-7H2O)

1512

Zinc Sulphate (ZnSO4-7H2O)

0.874

Other components

D-Glucose (Dextrose) 2000

Ethanolamine 1.9

Glutathione (reduced) 1

Phenol red 5

Sodium Pyruvate 110

Amino acids

Glycine 10

L-Alanine 8.9

L-Arginine 200

L-Asparagine 50

L-Aspartic Acid 20

L-Cystine 2HCI 65

L-Glutamic Acid 20

L-Histidine 15

L-Hydroxyproline 20

L-Isoleucine 50

L-Leucine 50

L-Lysine hydrochloride 40

L-Methionine 15

L-Phenylalanine 15

L-Proline 20

L-Serine 30

L-Theronine 20

L-Tryptophan 5

L-Tyrosine (disodium salt) 29

L-Valine 20

Vitamins

Ascorbic Acid Phosphate 2.5

Biotin 0.2

Choline chloride 3


Folic acid 1

i-Inositol 35

Niacinamide 1

Para-Aminobenzoic Acid 1

Pyridoxine hydrochoride 1

Riboflavin 0.2

Thiamine hydrochoride 1

Vitamin B12 0.005

Proteins

AlbuMAX® II 400

Human Transferrin (Holo) 7.5

Insulin Recombinant Full Chain

10

Reducing Agents

Glutathione, monosodium 1

Trace Elements

Ammonium Metavanadate 0.0003

Cupric Sulfate 0.00125

Manganous Chloride 0.00005

Sodium Selenite 0.005

Waymouth’s MediumBase cat. no. 31220


Inorganic salts

CaCl2 • H24O 120.00

KCl 150.00

KH2PO4O 80.00

MgCl2 • 6H2O 240.00

MgSO4 • 7H2O 200.00

NaCl 6000.00

NaHCO3 2240.00

Na2HPO4 (anhyd.) 300.00

Other components

D-Glucose 5000.00

Glutathione (reduced) 15.00

Hypoxanthine (sodium salt) 25.00

Phenol red 10.00

Amino acids



L-Cysteine HCl 90.00

L-Cystine 15.00


L-Glutamine 350.00

Glycine 50.00

L-Histidine (free base) 128.00

L-Isoleucine 25.00

L-Leucine 50.00


L-Methionine 50.00


L-Proline 50.00

L-Threonine 75.00

L-Tryptophan 40.00

L-Tyrosine 40.00

L-Valine 65.00

Vitamins

Ascorbic acid 17.50

Biotin 0.02



Folic Acid 0.40

i-Inositol 0.90

Niacinamide* 1.00

Pyridoxine HCl 1.00

Riboflavin 1.00

Thiamine HCl 10.00

Vitamin B12 0.20

1. Waymouth, C. (1968) Personal Communivation.* Also known as Nicotindamide.


edia3



Williams’ Medium E 1†

Base cat. no. 22551 32551Component 1X liquid, mg/L 1X liquid, mg/L

Inorganic salts

CaCl2 • 2H2O 264.00 264.00

CuSO4 • 5H2O 0.0001 0.0001

Fe(NO3)3 • 9H2O 0.0001 0.0001

KCl 400.00 400.00

MgSO4 • 7H2O 200.00 200.00

MnCl2 • 4H2O 0.0001 0.0001

NaCl 6800.00 6800.00

NaHCO3 2200.00 2200.00

NaH2PO4 • 2H2O 158.00 158.00

ZnSO4 • 7H2O 0.0002 0.0002

Other components

D-Glucose 2000.00 2000.00

Glutathione 0.05 0.05

Methyl Linoleate 0.03 0.03


Sodium Pyruvate 25.00 25.00

Amino acids

L-Alanine 90.00 90.00

L-Arginine 50.00 50.00

L-Asparagine • H2O 20.00 20.00

L-Aspartic Acid 30.00 30.00

L-Cysteine 40.00 40.00

L-Cystine 20.00 20.00

L-Ananyl-L-Glutamine – 434.00

L-Glutamic Acid 50.00 50.00

Glycine 50.00 50.00

L-Histidine 15.00 15.00


L-Leucine 75.00 75.00

L-Lysine HCla 87.00 87.00



L-Proline 30.00 30.00

L-Serine 10.00 10.00



L-Tyrosine 35.00 35.00

L-Valine 50.00 50.00

Vitamins

Ascorbic Acid 2.00 2.00

Biotin 0.50 0.50

D-Ca Pantothenate 1.00 1.00


Ergocalciferol 0.10 0.10



Menadione Sodium Bisulfate 0.01 0.01


Pyridoxal HCl 1.00 1.00


α-Tocopherol Phosphate, Disodium 0.01 0.01

Thiamine HCl 1.00 1.00

Vitamin A Acetate 0.10 0.10

Vitamin B12 0.20 0.20

1. Williams, G. M. And Gunn, J. M. (1974) Exp. Cell. Res., 89, 139. Supplement with 292.00 mg/L L-Glutamine (2mM)

a. Original formula lists Lisine at 70.00 mg/L* Also known as Nicotindamide.


80


Notes:


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Notes:


82


Notes:

Chapter 4 Serum-free and Specialty Media and ReagentsSummary of Serum-Free, Protein-free, and Chemically Defined Media . . . . . . . . . . . . . . . . . . . . . . 85

Summary of Specialty Media . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 85

Serum-free Media and Reagents . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 86

Differences Between Serum-free, Protein-free, and Chemically Defined Media . . . . . . . . . . . . . 86

Custom Formulations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 87

3D Matrices and Scaffolds . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 88

Expression Products . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 89

CHO Expression Products . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 89

Customized CHO Medium Products . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 90

DG44 Expression Products . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 91

293 Expression Products . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 93

Virus Production Medium Products . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 94

Viral Expression Products . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 97

Insect Cell Expression Products . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 98

Customized Insect Medium Products . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 101

Insect Medium Supplements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 103

Hybridoma and Monoclonal Antibody Products . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 106

Customized Hybridoma Medium Products . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 107

Cellular Studies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 109

Bronchial Epithelial Products . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 109

Dermatology Products . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 110

Hematology-Oncology Products . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 110

Hematopoiesis Products . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 113

Hepatocyte Products . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 113

Human Mammary Epithelial Products . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 114

Neurobiology Products . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 114

continued

83www .invitrogen .com

Stem Cell Research Products . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 117

B-27 Supplement . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 117

N-2 Supplement . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 117

Neurobasal™ Medium . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 117

Neurobasal™-A Medium . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 117

AIM V® Media (research grade) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 117

AIM V® Media (therapeutic grade) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 118

MesenPRO RS™ Medium . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 118

StemPro® hESC SFM . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 119

StemPro®-34 SFM . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 120

DMEM/F-12 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 120

KnocKout™ Serum Replacement . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 120

KnocKout™ DMEM . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 121

Geneticin® Selective Antibiotic . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 122

Blasticidin S HCI . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 123

Hygromycin B . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 123

GlutaMAX™-I Supplement . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 123

Fetal Bovine Serum, ES Cell-Qualified . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 123

CO2-Independent Medium . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 123

Basic Fibroblast Growth Factor (bFGF), Human, Recombinant . . . . . . . . . . . . . . . . . . . . . . . 124

Insulin-like Growth Factor-I (IGF-I), Human, Recombinant . . . . . . . . . . . . . . . . . . . . . . . . . . 124

Platelet Derived Growth Factor BB (PDGF BB), Human, Recombinant . . . . . . . . . . . . . . . . . . 124

Vascular-Endothelial Products . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 124

Cytogenetics Products . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 126

MarrowMAX™ . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 126

AmnioMAX™ . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 127

KaryoMAX® . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 127

Related Products for Cytogenetics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 128

Overview of Transfection, Selection, and Protein Delivery Methods . . . . . . . . . . . . . . . . . . . . . . 131

Transfection Reagents . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 132

Lipofectamine™ 2000 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 132

Lipofectamine™ LTX . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 132

FreeStyle™ MAX Reagent . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 132

293fectin™ Transfection Reagent . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 133

Lipofectamine™ RNAiMAX Transfection Reagent . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 133

Cellfectin® Transfection Reagent . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 133

PLUS™ Reagent . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 133

Lipofectamine™ Transfection Reagent . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 133

Lipofectamine™ 2000 Transfection Reagent . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 133

Chapter 4 Serum-free and Specialty Media and Reagents

84

Important Licensing Information: The products in this catalogue may covered by one or more Limited Use Label Licenses (see the Invitrogen website www .invitrogen .com) . By use of these products, you accept the terms and conditions of all applicable Limited Use Label Licenses . All products are for research use only, and are not intended for human or animal diagnostics, therapeutic, commercial, or other clinical uses unless otherwise specifically indicated in the applicable product Limited Use Label License(s) .

Summary of Serum-free, Protein-free, and Chemically Defined MediaApplication—Recombinant protein production Cells Media

Mammalian CHO, DG44 CD OptiCHO™ Medium, CD CHO Medium, CD CHO AGT™, CHO-S-SFM II

HeLa, HEK 293-F, HEK 293-H CD 293 Medium, CD 293 AGT™, 293 SFM II , Protein Expression Medium (PEM)

Insect Sf9, Sf21, Tn-368 Sf-900™ III, SFM Sf-900™ II SFM

BTI-TN-5B1-4 (High Five™) Express Five® SFM

Monoclonal antibody production Hybridomas, myelomas, NS0, NS1, Sp2 CD Hybridoma Medium, CD Hybridoma AGT™ Hybridoma-SFM PFHM-II Protein-free Hybridoma Medium

Application—Virus production

VERO, BHK-21, HEp-2, COS-7L VP-SFM, VP-SFM AGT™

HEK 293-F, HEK 293-H, HeLa CD 293 Medium, CD 293 AGT™ 293 SFM II

MDBK, MDCK, BHK-21, PK-15 OptiPRO™ SFM, Opti-MEM®

PER .C6® Protein Expression Medium (PEM), Adenovirus Expression Medium (AEM)

Application—Cellular studies

Dermatology Keratinocytes Defined Keratinocyte-SFM (without BPE) Keratinocyte-SFM (with BPE)

Hematology–oncology Lymphocytes (human primary lymphoid cells and transformed lymphoid lines)

AIM V® Media

Hematopoiesis Hematopoietic cells (bone marrow, peripheral blood, and neonatal cord blood)

StemPro®-34 SFM

Peripheral macrophages and monocytes Macrophage-SFM

Vascular research Endothelial cells Human Endothelial-SFM

Hepatic research Hepatocytes Hepatozyme-SFM

Neurobiology Neuronal cells Neurobasal™ Media, B-27 supplements

Transgenic mice Embryonic stem cells (mouse) KnocKout™ Serum Replacement KnocKout™ DMEM

For additional information on applications of serum-free media, see Appendix I, starting on page 187 .

Summary of Specialty Media* Cell type Specialty media Application

Amniocytes, chorionic villus cells AmnioMAX™-II Complete Medium In vitro propagation of primary human amniotic fluid cells for karyotyping

AmnioMAX™-C100 Complete Medium (basal medium and supplement)

In vitro propagation of primary human amniotic fluid cells for karyotyping

Supplement contains growth supplements (serum) and antibiotics

Lymphocytes PB-MAX™ Karyotyping Medium Fully supplemented (including FBS) RPMI-based medium for short-term cultivation of peripheral blood lymphocytes

Bone marrow and peripheral blood MarrowMAX™ Bone Marrow Medium A fully supplemented medium developed specifically to support bone marrow and peripheral blood cells in culture for in vitro cytogenetic analysis of hematological disease

Human mammary epithelial cells HuMEC Medium Optimized specifically for the growth of human mammary epithelial cells

Bronchial epithelial cells LHC Medium Engineered specifically for the growth of bronchial epithelial cells such as normal human bronchial epithelial cells and bovine bronchial epithelial cells without further supplements

* Specialty media are optimized and designed for specific applications and require serum supplementation . For additional information on applications of specialty media, see Appendix I, starting on page 196 .

gibco® cell culture catalogueSerum

-free and Specialty Media and Reagents

4



Serum-free Media and ReagentsFor information on classifications, production, or preparation of these products, see page 7 .

Boost Productivity, Meet Regulatory Requirements, and Simplify PurificationResearchers who “think downstream” choose gibco® products for serum-free cell culture .

If you’re working with cells that may ultimately be used in genomic, proteomic, biopharmaceutical, therapeutic, or diagnostic applications, your choice of culture media becomes more critical than ever .

Gibco® media meet pharmaceutical standards for quality, and are the most approved in the biopharmaceutical industry because they:

Minimize variables ■

Maximize yield ■

Simplify purification ■

Meet regulatory requirements ■

Optimize results ■

Optimize serum-free cell culture with gibco® productsChoose from chemically defined, protein-free, and serum-free ■

formulationsMinimize risk with animal origin–free media and reagents ■

Save time with preadapted cells and cell line development kits ■

Use liquid or Advanced Granulation Technology™ (AGT™) formats that ■

are scalable to large-volume cultures

Differences Between Serum-free, Protein-free, and Chemically Defined MediaSerum-free mediaGibco® Serum-free Media do not require supplementation with serum, but may contain discrete proteins or bulk protein fractions .

Protein-free mediaGibco® protein-free media contain no proteins, but may contain plant or yeast hydrolysates . Many are animal origin–free .

A N I M A LO R I G I N

FREE Chemically defined mediaGibco® chemically defined media contain no proteins, hydrolysates, peptides, or components of unknown composition . These media are animal origin–free, and all components have a known chemical structure .

Completely defined system eliminates variability ■

Consistent performance improves reproducibility ■

Decrease possibility of contamination by adventitious agents ■

Save time with simplified purification and downstream processing ■

Minimize risk with animal origin–free media and reagentsMaterials of animal origin have the potential to introduce adventitious agents into a cell culture system .

At Life Technologiestm we have developed a wide range of cell culture products that are completely free of animal-origin materials, by taking multiple steps to ensure the quality, safety, consistency, and regulatory compliance of our products:

Sourcing non–animal origin raw materials whenever possible ■

Substituting bioactive synthetic chemicals for their native ■

animal-derived counterpartsEnsuring solid documentation that details source, origin, and ■

manufacturing process for all raw materialsUsing non-protein compounds instead of insulin and transferrin in ■

protein-free and chemically defined mediaGibco® animal origin–free products do not contain material directly ■

derived from animal tissues, cells, or body fluids of higher eukaryotic organisms such as mammals (including humans), fish, birds, insects, etc . The term “animal origin” does not pertain to lower eukaryotic organisms such as the higher plants, fungi, protozoa, and algae, nor does it include prokaryotic organisms such as bacteria or blue-green algae .


86


Custom FormulationsAll serum-free formulations can be customized to meet your specifications . Our Custom Products Services group will work with you to determine composition, processing, packaging, labeling, shipping and storage conditions, and pricing for your formulation .

Limited-edition stocked customsSome formulations or supplements either in development or that have been requested as a custom by several customers may be offered as limited-edition stocked customs . This means we will have some made and ready to ship at your request . To learn more about additional options currently available, go to www .invitrogen .com/stockedcustoms .

Production, Preparation, and Quality ControlAll serum-free formulations are produced and prepared to the same quality standards that apply to all our gibco® products . For information on medium production, preparation, product packaging, closures, quality control procedures, and storage and sterilization, see page 7 .

For general outlines of methods of adaptation of cultures to serum-free conditions, direct and sequential (weaning), see Appendix I, page 187 .



4



3D Matrices and Scaffolds Geltrex™ matrix and the AlgiMatrix™ 3D Culture System contribute to a broader product portfolio of gibco ® 3D culture systems, which were developed to meet the demand for 3D cell culture products in fields such as stem cell and cancer research, drug development, toxicology, and development and morphogenesis, as well as tissue and organ engineering .

Geltrex™ Reduced Growth Factor Basement Membrane MatrixGeltrex™ matrix is a soluble form of reduced growth factor (RGF) basement membrane extract (BME) purified from continuous sheets of specialized extracellular matrix that form an interface between Engelbreth-Holm-Swarm (EHS) tumor cells . The major components of Geltrex™ matrix include laminin, collagen IV, entactin, and heparin sulfate proteoglycan, which provide the foundation for three-dimensional (3D) culture studies . Like naturally occurring basement membrane, Geltrex™ reduced growth factor basement membrane not only supports cells and cell layers, but also plays an essential role in tissue organization that affects cell adhesion, migration, proliferation, and differentiation .

It is easy to prepare suspensions using Geltrex™ matrix, and it offers, on average, a 2-fold reduction in several growth factors . As a result, analyses of specific growth factor effects in applications requiring basement membrane extract can be carried out in a controlled and reproducible way using Geltrex™ matrix .Recommended storage conditions: –20°C to –80°C .


1 ml 12760-013

5 ml 12760-021

Related products PageHuMEC-SFM . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 38KnocKout™ Serum Replacement . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 44Phosphate-Buffered Saline (PBS) 7 .2 (1X) . . . . . . . . . . . . . . . . . . . . . . . . . . . .125TrypLE™ Express Stable Trypsin

Replacement Enzyme without phenol red . . . . . . . . . . . . . . . . . . . . . .162Versene 1:5,000 (1X) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .163Visit us at www .invitrogen .com/3D-CellCulture to learn more about the Geltrex™ 3D Culture System and related products for 3D cell culture .

Collagen I, Rat TailCollagen I, Rat Tail is the most common fibrillar collagen (90%) found in skin, bone, tendons and other connective tissues . It is supplied at 5 mg/ml in a 20 ml vial providing the flexibility to dilute to lower concentrations . The detailed gelling procedure forms a firm, clear gel for 3D applications . Recommended storage conditions: 20°C to 80°C, in the dark .


20 ml A1048301

AlgiMatrix™ 3D Culture SystemThe AlgiMatrix™ 3D Culture System is a user-friendly non–animal-derived bio-scaffold for three-dimensional (3D) cell culture . The AlgiMatrix™ 3D Culture System creates a cell culture model system that more closely reflects normal cell morphology and behavior, to meet researchers’ requirements for 3D cell culture in fields such as toxicology, drug development, cancer and stem cell research, development and morphogenesis, tissue and organ engineering, heart disease, and diabetes .

The AlgiMatrix™ 3D Culture System is supplied in a 96-well format that fits well into the workflows of both lower- and higher-throughput users . Scientists can inoculate cells directly into the sterile microtiter plates preloaded with lyophilized alginate sponge, with no pretreatment or preparation required . The combination of convenience, high capacity, and microscopic visualization makes the AlgiMatrix™ 3D Culture System an ideal solution for broad basic research and drug discovery applications .

Other commercially available 3D matrices—including agar, collagen, and Matrigel™—are either difficult to use or are of animal origin, with significant lot-to-lot variability that can compromise performance . The AlgiMatrix™ sponge is formulated using pharmaceutical-grade raw material from brown seaweed . Its pure, chemically defined, and nontoxic macroporous structure yields improved lot-to-lot consistency, superior cell loading, and excellent nutrient delivery, without damage to cells . Recommended storage conditions: room temperature .


1 × 96-well plate 12684-015

5 × 96-well plates 12684-031

Related products PageDefined Keratinocyte-SFM System . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .110Hepatozyme-SFM . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .113KnocKout™ Serum Replacement . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .120Phosphate-Buffered Saline (PBS) 7 .2 (1X) . . . . . . . . . . . . . . . . . . . . . . . . . . . .141TrypLE™ Express Stable Trypsin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .160Versene 1:5,000 (1X) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .161

Visit www .invitrogen .com/3D-CellCulture to learn more about the AlgiMatrix™ 3D Culture System and related products for 3D cell culture .


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FREE FreeStyle™ MAX CHO Expression SystemThe FreeStyle™ MAX CHO Expression System is a completely animal origin–free protein expression system that provides scalable large-scale transient transfection and protein production in suspension FreeStyle™ CHO-S cells . FreeStyle™ CHO-S cells are Chinese hamster ovary (CHO) cells that have been adapted to serum-free suspension culture in FreeStyle™ CHO Expression Medium . Transfection and expression experiments may be performed directly in FreeStyle™ CHO Expression Medium without the need to change medium . The complete FreeStyle™ MAX CHO Expression System provides enough reagents to perform 25 transfections and one control transfection in a 30 ml volume, but larger-volume transfections may be performed using simple scale-up of reagents . At scale, the FreeStyle™ MAX CHO Expression System can provide milligram quantities of proteins in days instead of months .

The FreeStyle™ MAX CHO Expression System includes the following major components:

FreeStyle™ MAX Reagent—This transfection reagent provides high ■

transfection efficiency in suspension FreeStyle™ CHO-S cells .FreeStyle™ CHO-S cells—This cell line is adapted to high-density, ■

serum-free suspension culture in FreeStyle™ CHO Expression Medium and is capable of producing high levels of recombinant protein . CHO cells provide stable and accurate glycosylation of proteins for downstream preclinical and therapeutic applications .FreeStyle™ CHO Expression Medium—This is a defined, serum-free ■

medium formulated specifically to allow growth and large-scale transfection of suspension FreeStyle™ CHO-S cells .OptiPRO™ Serum-free Medium to facilitate optimal formation of ■

DNA-lipid complexes .Recommended storage conditions : FreeStyle™ CHO cells, liquid nitrogen; FreeStyle™ MAX Reagent, 4°C, do not freeze; FreeStyle™ CHO Expression Medium and OptiPRO™ Serum-free Medium, 4°C, in the dark; pCMV•SPORT-β-gal control vector, –20°C .


1 kit K9000-20

Transient transfection using FreeStyle™ MAX Reagent with FreeStyle™ CHO cells in a 10 L Wave Bioreactor® for antibody production.

Expression ProductsCHO Expression Products


FREE CD OptiCHO™ MediumChemically defined (CD) medium optimized for recombinant CHO cells .

CD OptiCHO™ Medium is specifically designed to offer high performance and yield with recombinant CHO cells in a chemically defined environment . The protein-free, animal origin–free formulation provides consistency and reliability and reduces the need to screen for adventitious agents .

Optimized to support and maximize recombinant CHO cell growth ■

and protein expression in suspension cultureProvides up to 40% greater growth and production than existing ■

media for selected cell lines and applicationsTruly chemically defined and protein-free formulation contains no ■

hydrolysates, improving lot-to-lot consistency and reliabilityRecommended storage conditions: 2°C to 8°C, in the dark . Guaranteed stable for 12 months when properly stored .


1,000 ml 12681-011

6 × 1,000 ml 12681-029

Optimized growth of CHO cells in CD OptiCHO™ Medium. Recombinant CHO cells were seeded in 30 ml of CD OptiCHO™ Medium or CD CHO Medium at a viable cell density of 4 × 105 cells/ml in a 125 ml Corning® shake flask . Cell density and viability were monitored daily using a Vi-CELL® XR Cell Counter (Beckman Coulter) . Recombinant protein (EPO) expression level was monitored by ELISA .

4 6 8 1020 12

CD OptiCHO™ (EPO)CD CHO (EPO)

CD OptiCHO™ (viable cell density)CD CHO (viable cell density)

EPO

(mg/

L)

0

Time (days)

Viab

le c

ell d

ensi

ty (c

ells

/ml)

1 × 107

1 × 106

300

100

200

400

Hum

an Ig

G y

ield

(mg/

L)

12118531

80

80

80

80

80

80

80

80

8010 L Wave transfection

50 ml control transfectionin a shake �ask

1.5

9.812.5

37.9

29.6

56.451.2

60.1

67.570.1

66.662.2

Days after transfection



4




FREE CD CHO Medium (1X), liquidCD CHO Medium is a chemically defined, protein-free medium optimized for the growth of Chinese hamster ovary (CHO) cells and expression of recombinant proteins in suspension culture . CD CHO Medium contains no proteins or peptide components of animal, plant, or synthetic origin . There are also no undefined lysates or hydrolysates in the formulation .

CD CHO Medium is made without hypoxanthine and thymidine for ■

use in dihydrofolate reductase (DHFR) amplified systems; for other applications, add 10 ml per liter of HT Supplement (Cat . no . 11067-030) prior to useFormulated without L-glutamine for increased stability; add 40 ml of ■

200 mM L-glutamine per 1,000 ml of medium prior to useRecommended storage conditions: 2°C to 8°C, in the dark .Notes: A CHO Medium Master File has been submitted to the FDA . Permission to cross-reference the Master File may be obtained by contacting Invitrogen Technical Support or your local Sales Representative .


500 ml 10743-011

1,000 ml 10743-029

Contains no glutamine, hypoxanthine, thymidine, or phenol red .

Related products PageL-Glutamine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .149GlutaMAX™-I Supplement . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .123HT Supplement . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .107CHO-S Cells adapted to CD CHO Medium . . . . . . . . . . . . . . . . . . . . . . . . . .178DG44 Cells adapted to DG44 Medium . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .178


FREE CD CHO AGT™CD CHO AGT™ is a chemically defined, protein-free medium in a new easy-to-use granular format . CD CHO AGT™ is optimized for the growth of Chinese hamster ovary (CHO) cells and expression of recombinant proteins in suspension culture .Recommended storage conditions: 2°C to 8°C, in the dark .


1 × 1 L 12490-017

1 × 10 L 12490-025

Related products PageL-Glutamine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .149GlutaMAX™-I Supplement . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .123HT Supplement . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .107CHO-S Cells adapted to CD CHO Medium . . . . . . . . . . . . . . . . . . . . . . . . . .178DG44 Cells adapted to DG44 Medium . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .178

CHO-S-SFM IICHO-S-SFM II is a complete, serum-free, low-protein (less than 100 µg/ml) medium optimized for the growth and maintenance of Chinese hamster ovary (CHO) cells in suspension culture and for the production of recombinant proteins expressed by CHO cells .1,2 This ready-to-use medium is suitable for the suspension culturing of CHO and other cells in batch, continuous, and perfusion culture systems .Recommended storage conditions: 2°C to 8°C, in the dark .Notes: A CHO Medium Master File has been submitted to the FDA . Permission to cross-reference the Master File may be obtained by contacting Invitrogen Technical Support or your local Sales Representative .

References1 . Tilkins, M .L ., Judd, D .A ., Weiss, S .A ., and Gorfien, S .F . (1992) FOCUS® 14, 95 . 2 . Battista, P .J ., Tilkins, M .L ., Judd, D .A ., Godwin, G .P ., and Gorfien, S .F . (1994) American

Biotechnology Laboratory 12:5, 64 .


500 ml 12052-114

1,000 ml 12052-098

Contains L-glutamine .

Quantity Cat. no.

500 ml 31033-020

Contains L-glutamine, but no hypoxanthine or thymidine .

Customized CHO Medium ProductsWe offer several CHO formulations that are not routinely stocked in inventory, but are available on a make-to-order basis . We realize that not all needs are alike, so we’ve developed the capabilities to provide the desired configuration (formulation and packaging) of product type, as well as the operational systems that can provide these products in a timely fashion . Contact our Custom Products Services group for assistance in selecting the product that best meets your needs

Customized products are available in a wide array of packaging, from standard PET bottles (100 ml, 500 ml, and 1,000 ml) to custom media bags (1 L, 5 L, 10 L, and 200 L) .

We offer the following on a custom basis:Adherent CHO-SFM: designed for anchorage-dependent CHO systems . CHO-III-PFM: devoid of animal-sourced raw materials, this essentially protein-free medium contains a vegetable hydrolysate for added perform ance .Notes: A CHO-III-PFM Master File has been submitted to the FDA . Permission to cross-reference the Master File may be obtained by contacting Invitrogen Technical Support or your local Sales Representative . Please note that variations of the above products, such classical media as DMEM/F-12, Nutrient Mixture F-12, and MCDB 301, as well as your own in-house developed formulations, can be tailored to your specific selection/amplification system (DHFR, glutamine synthetase, neomycin resistance, etc .) .


90


CHO CD EfficientFeed™ KitThe CHO CD EfficientFeed™ Kit is a system designed to help you more quickly and efficiently identify a feed supplement formulation that will improve fed-batch productivity 2- to 5-fold versus batch conditions, as well as identify a feed supplement formulation that will help develop bioreactor cell seed stocks more quickly . The kit contains feed supple-ments A and B, based on successful chemically-defined media formula-tions you may be using or considering for your batch culture medium (CD OptiCHO™ Medium or CD CHO Medium .)Recommended storage conditions: 2°C to 8°C, in the dark .


CHO CD EfficientFeed™ Kit 1 kit A10241-01

Kit components also available separately

CHO CD EfficientFeed™ A 1000ml A10234-01

CHO CD EfficientFeed™ B 1000ml A10240-01

DG44 Expression Products

DG44 Cells and Media KitThe DG44 Cells and Media Kit are optimized for the growth of dihydrofolate reductase–deficient (DHFR–) Chinese hamster ovary (CHO) cells and expression of recombinant proteins in suspension culture . The kit provides CD DG44 Medium, a chemically defined, protein-free medium specifically designed to enable optimal growth and expression of DG44 cells in suspension culture, and DG44 cells preadapted to CD DG44 Medium . Cells are selected for superior cell growth and transfection efficiencies . In addition, the DG44 Cells and Media Kit includes L-glutamine and Pluronic® F-68 for optimal growth and protein expression of DG44 cells in a protein-free, chemically defined environment . The cells are banked under cGMP conditions and with full documentation available with commercial use license .Recommended storage conditions: CD DG44 Medium, 2°C to 8°C, in the dark; DG44 cells, in liquid nitrogen; L-glutamine, –5°C to –20°C; Pluronic® F-68, 15°C to 30°C .Notes: A CHO Medium Master File has been submitted to the FDA . Permission to cross-reference the Master File may be obtained by contac t ing I nv i t rogen Technica l Suppor t o r your loca l Sales Representative .

Pluronic® is a registered trademark of BASF Corporation .


1 Kit A1100-001



4



OptiCHO™ Protein Express Kit The OptiCHO™ Protein Express Kit is designed for faster serum-free cell line development and cloning for single or two sub unit protein expression in suspension culture in dihydrofolate reductase-deficient (DHFR-) Chinese hamster ovary (CHO) DG44 cells created under cGMP condiitons .The Kit provides a complete workflow protocol and manual information provided on a reuseable USB flash drive, as well as the necessary components for efficient transfection and cloning of DHFR-DG44 cells in a serum-free en-vironments and subsequent development of a stable cell line for protein production .

The Kit contains:Two expression vectors for light and heavy chains-the pOptiVEC™- ■

TOPO® bicistronic cloning vector and pcDNA™ 3 .3 TOPO® cloning vector, the vectors are TOPO adapted to enable cloning of a PCR product containing your gene of interest with >85% efficiency .FreeStyle™ MAX Reagent ■ —an animal origin free transfection reagent offering high transfection efficiencies and consistent results .CD OptiCHO™ Medium ■ —a chemically defined, protein free medium (without hypoxanthine or thymidine) specially designed for high-density suspension of culture of recombinant CHO DG44 cells and secreted protein yield .CD DG44 Medium ■ —a chemically defined, protein free medium supplemented with hypoxanthine and thymidine to support optimal growth of DG44 cells in suspension culture .DG44 cells ■ —(banked under cGMP conditions and with full documentat ion avai lable with commercial use l icense) – preadapted to CD DG44and selected for superior cell growth and transfection efficiencies .


OptiCHO™ Protein Express Kit 1 kit A1099901

Kit components, also available separately

pOptiVEC™-TOPO® TA Cloning Kit 1 kit 12744-017

CD DG44 Medium 1,000 ml 12610-010

FreeStyle™ MAX Reagent 1 ml 16447-100

OptiPRO™ SFM 100 ml 12309-050

CD OptiCHO™ Medium 1,000 ml 12681-011

Pluronic® F-68, 10% 100 ml 24040-032

L-glutamine-200 mM (100X) 100 ml 25030-081



92


293 Expression Products

CD 293 Medium (1X), liquidCD 293 Medium is a chemically defined, protein-free medium optimized for the growth of suspension cultures of 293 cells . CD 293 Medium contains no proteins or peptides of animal, plant, or synthetic origin . There are also no undefined lysates or hydrolysates in the formulation .

Formulated without L-glutamine for increased stability; add 20 ml of 200 mM L-glutamine or 20 ml of GlutaMAX™-I Supplement per 1,000 ml of medium prior to use .Notes: A 293 Medium Master file has been submitted to the FDA . If you wish to cross-reference a Master file for CD 293 Medium, please contact Invitrogen Technical Support or your local Sales Representative .Recommended storage conditions: 2°C to 8°C, in the dark .


1,000 ml 11913-019

Related products PageL-Glutamine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .149GlutaMAX™-I Supplement . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .123293-F Cells adapted to CD 293 Medium . . . . . . . . . . . . . . . . . . . . . . . . . . . .177293-H Cells adapted to CD 293 Medium . . . . . . . . . . . . . . . . . . . . . . . . . . . .177

CD 293 AGT™CD 293 AGT™ is a chemically defined, protein-free medium in a new easy-to-use granular format . CD 293 AGT™ is optimized for the growth of suspension cultures of 293 cells .Recommended storage conditions: 2°C to 8°C, in the dark .


1 × 1 L 12529-020

1 × 10 L 12529-012

293 SFM II (1X), liquid293 SFM II is a serum-free, low-protein (<10 µg/ml) formulation optimized for high-density suspension culture of 293 cells .1 (It is not suitable for adherent 293 cell cultures .) This product replaces 293 SFM (Cat . no . 10970-010) . 293 SFM II is animal origin–free .

Formulated without L-glutamine for increased stability; add 20 ml of 200 mM L-glutamine or 20 ml of GlutaMAX™-I Supplement per 1,000 ml of medium prior to use .Notes: A 293 Medium Master file has been submitted to the FDA . Permission to cross-reference the Master File may be obtained by contacting Invitrogen Technical Support or your local Sales Representative .Recommended storage conditions: 2°C to 8°C, in the dark .

Reference1 . Epstein, D .A ., Godwin, G .P ., Gruber, D .F ., and Grefrath, P .L . (1999) FOCUS® 21, 22 .


1,000 ml 11686-029


FreeStyle™ 293 Expression MediumFreeStyle™ 293 Expression Medium is a chemically defined, protein-free medium specifically developed for the ability to support the growth and transfection of 293-F cells under suspension-type culture conditions . The medium is a complete, ready-to-use medium that has been supplemented with GlutaMAX™-I Supplement and is animal origin–free . FreeStyle™ 293 Expression Medium is able to save significant time and costs associated with adaptation of cell cultures to serum-free conditions .Recommended storage conditions: 2°C to 8°C, in the dark .

References1 . Graham, F .L ., Smiley, J ., Russell, W .C ., and Nairn, R . (1977) J. Gen. Virol. 36, 59 .2 . Harrison, T ., Graham, F ., and Williams, J . (1977) Virology 77, 319 .3 . Ciccarone, V ., Chu, Y ., Schifferli, K ., Pichet, J .P ., Hawley-Nelson, P ., Evans, K ., Roy,

L ., and Bennett, S . (1999) FOCUS® 21, 54 .4 . Epstein, D .A ., Godwin, G .P ., Gruber, D .F ., and Grefrath, P .I . (1999) FOCUS® 21, 22 .


1,000 ml 12338-018

6 × 1,000 ml (case) 12338-026

8 L fill in 20 L Wave Cellbag® 0070049BB



4



FreeStyle™ 293 Expression SystemThe FreeStyle™ 293 Expression System simplifies and streamlines production of protein in 293 cells . Using the FreeStyle™ 293 Expression System, you can transfect 293 cells in suspension, eliminating the need to carry multiple flasks of adherent cells when large numbers of cells are required . The FreeStyle™ 293 Expression System offers the following key components to optimize transfection and expression in 293 cells:

293fectin™ Reagent— ■ a transfection reagent for the highest transfec-tion efficiency in suspension 293 cellsGibco® FreeStyle™ 293 Expression Medium— ■ a chemically defined, protein-free medium specifically designed to enable efficient transfection and optimal growth of 293 cells in suspensionFreeStyle™ 293-F cells— ■ a clone of 293 cells with superior handling properties in FreeStyle™ 293 Expression MediumFreeStyle™ 293-F cells, 293fectin™ Reagent, and gibco® FreeStyle™

293 Expression Medium work together to give you the most robust production of bioactive protein possible .

In addition, the FreeStyle™ 293 Expression System provides Gibco® Opti-MEM® I for improved DNA-lipid complex formation and a β-galactosidase expression plasmid to test transfection efficiency . The FreeStyle™ 293 Expression System contains enough reagents to perform twenty-five 30 ml transfections, but is designed to be scalable so that other transfection volumes can easily be used if required .Recommended storage conditions: 293fectin™, gibco® FreeStyle™ 293 Expression Medium, and Gibco® Opti-MEM®, 4°C; pCMV•SPORT-β-gal, –20°C; FreeStyle™ 293-F cells, in liquid nitrogen; gibco® FreeStyle™ 293 Expression Medium, in the dark .


FreeStyle™ 293 Expression Kit 1 kit K9000-01

GIBCO® FreeStyle™ 293 Expression Medium 1,000 ml 12338-018

293fectin™ Transfection Reagent 1 ml 12347-019

FreeStyle™ 293-F Cells 1 × 107 cells R790-07

Customized 293 Expression Medium Products293 Expression Medium products and your in-house formulations are available on a make-to-order basis . We realize that not all needs are alike, so we’ve developed the capabilities to provide the desired configuration (formulation and packaging) of product type, as well as the operational systems that can provide these products in a timely fashion . Please contact our Custom Products Services group for assistance in selecting the products and packaging that best meet your requirements .

Virus Production Medium Products


FREE OptiPRO™ SFM (1X), liquidOptiPRO™ SFM is an animal origin–free serum-free medium designed for growth of several kidney-derived cell lines including MDCK, MDBK, VERO, BHK-21, and PK-15, which are important for virus and recombinant protein production .Recommended storage conditions: 2°C to 8°C, in the dark .Notes: An OptiPRO™ SFM Master File has been submitted to the FDA . Permission to cross-reference the Master File may be obtained by contacting Invitrogen Technical Support or your local Sales Representative .

This product is the subject of US Patent No . 6,767,741 .


1,000 ml 12309-019

Opti-MEM® I Reduced-Serum Medium (1X), liquid (with GlutaMAX™-I)A GlutaMAX™-I medium . Recommended storage conditions: 2°C to 8°C, in the dark .Intended use: In vitro diagnostic (IVD) .


500 ml 51985-026

10 × 500 ml (case) 51985-042

Contains GlutaMAX™-I substituted on a molar equivalent basis for L-glutamine, HEPES buffer, 2,400 mg/L sodium bicarbonate, hypoxanthine, thymidine, sodium pyruvate, trace elements, growth factors, and phenol red reduced to 1 .1 mg/L .

Opti-MEM® I Reduced-Serum Medium (1X), liquidRecommended storage conditions: 2°C to 8°C, in the dark .Intended use: In vitro diagnostic (IVD) .


100 ml 31985-062

500 ml 31985-047

10 × 500 ml (case) 31985-054

Contains HEPES buffer, 2,400 mg/L sodium bicarbonate, hypoxanthine, thymidine, sodium pyruvate, L-glutamine, trace elements, growth factors, and phenol red reduced to 1 .1 mg/L .


94


Opti-MEM® I Reduced-Serum Medium (1X), liquid (without phenol red)Recommended storage conditions: 2°C to 8°C, in the dark .


500 ml 11058-021

Contains HEPES buffer, 2,400 mg/L sodium bicarbonate, hypoxanthine, thymidine, sodium pyruvate, L-glutamine, trace elements, and growth factors, but no phenol red .

Opti-MEM® I Reduced-Serum Medium, powderA modification of MEM (Eagle’s) . Recommended storage conditions: 2°C to 8°C, in the dark .Intended use: In vitro diagnostic (IVD) .See page 84 for procedure to prepare powdered Opti-MEM® I, 10X .


10 × 1 L 22600-050

1 × 10 L 22600-134

Contains HEPES buffer, hypo xanthine, thymidine, sodium pyruvate, L-glutamine, trace elements, growth factors, and phenol red reduced to 1 .1 mg/L, but no sodium bicarbonate .


FREE VP-SFM (1X), liquidVP-SFM is a serum-free, ultralow-protein (5 µg/ml) medium containing no components of animal or human origin . It is designed for the growth of VERO cells for virus production .1 This medium is suitable for the growth of COS-7, MDCK, BHK-21 (suspension culture), and HEp-2 cells, and for recombinant protein production .

Formulated without L-glutamine for increased stability; add 20 ml of 200 mM L-glutamine or 20 ml of GlutaMAX®-I Supplement per 1,000 ml of medium prior to use .Recommended storage conditions: 2°C to 8°C, in the dark .Notes: A VP-SFM Master File has been submitted to the FDA . Permission to cross-reference the Master File may be obtained by contacting Invitrogen Technical Support or your local Sales Representative .

Reference1 . Price, P . and Evege, E . (1997) FOCUS® 19, 67 .

This product is the subject of US Patent No . 6,103,529 .


1,000 ml 11681-020

Related products PageL-Glutamine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .149GlutaMAX™-I Supplement . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .123


FREE VP-SFM AGT™VP-SFM AGT™ is a serum-free, ultralow-protein (5 µg/ml) medium in a new easy-to-use granular format . VP-SFM AGT™ is designed for the growth of VERO cells for virus production . It is also suitable for the growth of COS-7, MDCK, BHK-21, and HEp-2 cells and for recombinant protein production .Recommended storage conditions: 2°C to 8°C, in the dark .


1 × 1 L 12559-027

1 × 10 L 12559-019

Related products PageL-Glutamine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .149GlutaMAX™-I Supplement . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .123Distilled Water . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .161



4



PER.C6® EpCAM cell growth and IgG production in PEM, VPRO, and AEM. PER .C6® EpCAM cells were adapted from DMEM/F-12 into gibco® PEM, SAFC/Sigma EX-CELL® VPRO Serum-free Medium for Retinoblast Cells, and gibco® AEM over several passages until stable cultures were achieved . Data represent growth experiments performed in triplicate for each condition . Cells grown in AEM and VPRO media were seeded at 3 × 105 vc/ml into 250 ml shake flasks with a working volume of 40 ml; cells grown in PEM were seeded at 1 × 105 vc/ml into 250 ml shake flasks with a working volume of 40 ml, as PEM supports more optimized conditions with a lower seeding density and extended growth .

Time (days)

0 1 2 3 4 5 6 7 8 9

Via

ble

cel

l den

sity

(vc/

ml)

IgG

(mg/

L)

1 × 107

1 × 106

1 × 105

200

400

600

PEM cell densityVPRO cell densityAEM cell densityPEM IgGVPRO IgGAEM IgG

0


FREE Protein Expression Medium (PEM)Efficient production of recombinant proteins in PER .C6® cells ■

Supports the large-scale, high-density culture of transfected PER .C6® ■

cells in shake flasks, roller bottles, and bioreactorsFormulated without L-glutamine for maximum stability ■

Supports parental PER .C6®, HEK 293, and HeLa cell growth in ■

suspension culture, as well as transfected PER .C6® cells for protein expression applications

Recommended storage conditions: 2°C to 8°C, in the dark .


1,000 ml 12661-013

Contains no components directly derived from human or animal sources and one low-level, recombinant protein, minimizing the opportunity to introduce adventi-tious agents .


FREE Adenovirus Expression Medium (AEM) Adenovirus Expression Medium (AEM) is recommended for the growth of PER .C6® cells in suspension and for the production of viruses and viral vectors in these cells . It is an animal origin–free cell culture medium without components directly derived from human or animal sources . Each lot of AEM is performance tested for cell growth with PER .C6® cells, and AEM has been qualified to produce adenovirus and influenza viruses . It also supports HEK 293 cell growth .

Glutamine or GlutaMAX™-I supplementation is required .Recommended storage conditions: 2°C to 8°C, in the dark .Notes: An Adenovirus Expression Medium Master File has been submitted to the FDA . Permission to cross-reference the Master File may be obtained by contacting Invitrogen Technical Support or your local Sales Representative .


1,000 ml 12582-011


PER .C6® is a registered trademark of Crucell Holland B .V .


FREE CD 293 Medium (1X), liquidCD 293 Medium is a chemically defined, protein-free medium optimized for the growth of suspension cultures of 293 cells . CD 293 Medium contains no proteins or peptides of animal, plant, or synthetic origin . There are also no undefined lysates or hydrolysates in the formulation .

Formulated without L-glutamine for increased stability; add 20 ml of 200 mM L-glutamine or 20 ml of GlutaMAX™-I Supplement per 1,000 ml of medium prior to use .Recommended storage conditions: 2°C to 8°C, in the dark .Notes: A 293 Medium Master file has been submitted to the FDA . If you wish to cross-reference a Master file for CD 293 Medium, please contact Invitrogen Technical Support or your local Sales Representative .


1,000 ml 11913-019



96



FREE CD 293 AGT™CD 293 AGT™ is a chemically defined, protein-free medium in a new easy-to-use granular format . CD 293 AGT™ is optimized for the growth of suspension cultures of 293 cells .Recommended storage conditions: 2°C to 8°C, in the dark .


1 × 1 L 12529-020

1 × 10 L 12529-012

Related products PageL-Glutamine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .149GlutaMAX™-I Supplement . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .123Distilled Water . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .163293-F Cells adapted to CD 293 Medium . . . . . . . . . . . . . . . . . . . . . . . . . . . .161293-H Cells adapted to CD 293 Medium . . . . . . . . . . . . . . . . . . . . . . . . . . . .177


FREE 293 SFM II (1X), liquid293 SFM II is an animal origin–free, serum-free, low-protein (<10 µg/ml) formulation optimized for high-density suspension culture of 293 cells .1 (It is not suitable for adherent 293 cell cultures .) This product replaces 293 SFM (Cat . no . 10970-010) .

It is formulated without L-glutamine for increased stability . Add 20 ml of 200 mM L-glutamine or 20 ml of GlutaMAX™-I Supplement per 1,000 ml of medium prior to use .Recommended storage conditions: 2°C to 8°C, in the dark .Notes: A 293 Medium Master File has been submitted to the FDA . Permission to cross-reference the Master File may be obtained by contacting Invitrogen Technical Support or your local Sales Representative .

Reference1 . Epstein, D .A ., Godwin, G .P ., Gruber, D .F ., and Grefrath, P .L . (1999) FOCUS® 21, 22 .


1,000 ml 11686-029


Customized Virus Production Medium ProductsPlease note that the virus production products and your in-house formulations are available on a make-to-order basis . We realize that not all needs are alike, so we’ve developed the capabilities to provide the desired configuration (formulation and packaging) of product type, as well as the operational systems that can provide these products in a timely fashion . Please contact our Custom Products Services group for assistance in selecting the products and packaging that best meet your requirements .

Viral Expression Products


FREE ViraPower™ Adenoviral Expression SystemThe ViraPower™ Adenoviral Expression System has improved the process of making an adenoviral construct . Get clones with less effort and go directly to your expression experiments .


ViraPower™ Adenoviral Gateway® Expression Kit* 1 kit K4930-00

pAd/BLOCK-iT™ Adenoviral RNAi Vector 6μg V492-20

BLOCK-iT™ U6 RNAi Entry Vector Kit 1 kit K4945-00

pAd/PL-DEST™ Gateway® Vector 6 µg V494-20

293A Cell Line 3 × 106 cells R705-07

* Kit contains Gateway® DEST Vector and 293A Cell Line .

For more information on how ViraPower™ adenoviral expression and Gateway® technology work, visit our website at www .invitrogen .com .



4



Overview of Baculovirus Expression SystemsExpression in insect cells offers significant advantages, including high expres-sion levels, ease of scale-up, production of proteins with posttranslational modifications, and simplified cell growth . Insect cells do not require CO2 for growth and can be readily adapted to high-density suspension culture for large-scale expression . Many of the posttranslational modification pathways present in mammalian systems are also utilized in insect cells, allowing the production of recombinant protein that is antigenically, immunogenically, and functionally similar to the native mammalian protein . Baculovirus expression systems are powerful and versatile systems for high-level recombinant protein expression in insect cells . Expression levels up to 500 mg/L have been reported using the baculovirus expres-sion system, making it an ideal system for high-level expression .The BaculoDirect™ Baculovirus Expression System is the fastest and easiest method available for generating recombinant baculo virus . Purified virus can be isolated within one week without plaque purification . The protein yield often depends on the nature of the gene . Yields of 25–250 µg/ml can be expected but are not guaranteed . The BaculoDirect™ Linear DNA includes attR sites for rapid and efficient recombinational cloning with a Gateway® entry clone . The resulting recombinant DNA can be taken directly from the LR reaction mix and used to transfect insect cells, saving a significant amount of time . Purified virus can be isolated within one week . The reduction of hands-on time makes the BaculoDirect™ system ideal for high-throughput expression .The Bac-to-Bac® Baculovirus Expression System uses a unique bacmid shuttle vector that recombines with an expression cassette by site-specific transposition to create an expression bacmid . The expression bacmid is then transfected into insect cells to generate recombinant baculovirus . The positive expression bacmid is selected by a simple blue/white screening in E. coli . Once confirmed, it is transfected into insect cells to generate recombinant baculovirus . No tedious plaque purification is needed .The Bac-N-Blue™ Baculovirus Expression System has been used for over a decade to produce high levels of recombinant proteins . Improvements to the AcMNPV DNA, higher-expressing cell lines, and small vectors have made this system faster and easier to use .

Insect Cell Expression Products

Overview of Stable Insect Expression SystemsStable insect expression systems combine the advantages of both baculovirus and mammalian expression . Stable insect systems are simple to use with straightforward techniques for transfection and selection similar to those used with mammalian expression systems . At the same time, stable insect systems often achieve higher levels of expression than mammalian systems and are particularly useful for production of secreted proteins . Like baculovirus systems, high levels of expression are attainable using stable insect expression systems . An added benefit is the reliability and reproducibility of expression levels . This differs from baculovirus expression levels that can vary from experiment to experiment . Once a stable cell line is produced, the expression levels are maintained over time . In addition, stable insect expression systems produce protein from healthy, logarithmically growing cells, resulting in high-quality protein .

The InsectSelect™ System is a stable insect expression system designed for continuous, non-lytic expression using Sf9, Sf21, or High Five™ cells . Stable expression with the InsectSelect™ system can be achieved in just three simple steps: 1 . Clone a gene of interest into an InsectSelect™ vector .2 . Transfect insect cells . 3 . Select stable clones . The InsectSelect™ System uses blasticidin

or Zeocin™ selection for efficient selection of stable clones . With blasticidin, clones can be established in as little as one week . The protein yield often depends on the nature of the gene . Yields of 1–10 µg/ml can be expected but are not guaranteed .

Insect Expression SystemsFusion partner

System Host Position Purification Epitope Promoter Advantage

BaculoDirect™ Sf9, Sf21, or High Five™

C-term 6xHis V5 Polyhedrin Easiest method of baculovirus production, ideal for HTP

Bac-to-Bac® Sf9, Sf21, or High Five™

N-term 6xHis NA Polyhedrin or p10

Rapid baculovirus production for small-scale expression

Bac-N-Blue™ Sf9, Sf21, or High Five™

N-term C-term

6xHis Xpress™ V5

Polyhedrin High-level expression, well-characterized system


98



FREE Sf-900™ III SFMSf-900™ III SFM is a protein-free, animal origin–free medium that provides consistency and reliability in cell growth and protein expression . This product is formulated with one-third of the hydrolysate found in Sf-900™ ll . Because this medium does not include animal-derived components, the need to screen for adventitious agents is reduced .

Animal origin–free medium with improved consistency in insect cell ■

cultureOptimized to support and maximize Sf9 and Sf21 cell growth and ■

recombinant gene expression in suspension culture using both baculovirus and stable insect expression systemsSupports Sf9 cell growth to densities of 10 to 14 × 10 ■ 6 cells/mlProtein-free, low-hydrolysate formulation improves lot-to-lot ■

consistency and reliabilityRecommended storage conditions: 2°C to 8°C, in the dark . Guaranteed stable for 12 months when properly stored .


500 ml 12658-019

1,000 ml 12658-027

10 L 12658-035

Contains no components of animal origin, facilitating the regulatory process .

Sf-900™ II SFMSf-900™ II SFM is a protein-free insect cell culture medium optimized for the growth and maintenance of Spodoptera frugiperda (Sf9 and Sf21) cells and for large-scale production of recombinant proteins expressed using the baculovirus expression vector system (BEVS) . This medium is suitable for suspension and monolayer culture methods and supports growth of other lepidopteran cell lines .1

Notes: An Sf-900™ II SFM Master File has been submitted to the FDA . Permission to cross-reference the Master File may be obtained by contacting Invitrogen Technical Support or your local Sales Representative .

Reference1 . Godwin, G .P . and Whitford, W . (1993) FOCUS® 15, 44 .

Sf-900™ II SFM (1X), liquidInsect cell culture tested .Recommended storage conditions: 2°C to 8°C, in the dark .


500 ml 10902-096

1,000 ml 10902-088

6 × 1,000 ml (case) 10902-104

5 L 10902-161

20 L 10902-187

10 L fill in 20 L Wave Cellbag® 0070037BA


Related products PageSf21 Cells adapted to Sf-900™ II SFM . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .178Sf9 Cells adapted to Sf-900™ II SFM . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .177

Sf-900™ II SFM (1X), liquid (without Met and Cystine)Insect cell culture tested .Recommended storage conditions: 2°C to 8°C, in the dark .


500 ml 21012-026

Contains L-glutamine, but no L-methionine or L-cystine .

Related products PageSf21 Cells adapted to Sf-900™ II SFM . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .175Sf9 Cells adapted to Sf-900™ II SFM . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .175

Sf-900™ Medium (1.3X), liquidRecommended storage conditions: 2°C to 8°C, in the dark .

Sf9 cell growth in Sf-900™ II SFM and Sf-900™ III SFM at passage 6. Sf9 cells were maintained in Sf-900™ II SFM or Sf-900™ III SFM media . After 6 passages in the test media, cell growth curves were determined by seeding 30 ml cultures in 125 ml shake flasks at 500,000 cells/ml, and counting viable cell number on each day post-seed . Data points represent the averages of 3 replicate flasks, and the error bars denote one standard deviation .

Day 6Day 5Day 4 Day 7

Cel

ls/m

l

14 × 106

16 × 106

12 × 106

10 × 106

8 × 106

6 × 106

0

2 × 106

4 × 106

Sf-900™ II, lot #1

Sf-900™ II, lot #2

Sf-900™ III, lot #1





4




100 ml 10967-032


Schneider’s Drosophila Medium (1X), liquidModified and insect cell culture tested .Recommended storage conditions: 2°C to 8°C .


500 ml 21720-024

Express Five® SFM (1X), liquidExpress Five® SFM is a protein-free insect cell culture medium optimized to support maximum cell growth and recombinant gene expression by the baculovirus expression vector system (BEVS) in the BTI-TN-5B1-4 (High Five™) Trichoplusia ni cell line . This unique medium eliminates problems associated with the culturing of the High Five™ cell line . It is optimized for serum-free growth in monolayer and suspension culture, boosting insect cell growth to densities exceeding 5 × 106 cells/ml and population doubling times of 16 to 30 hours . It demonstrates expression of recombinant proteins that is equal or superior to that of other serum-free formulations . It is formulated without L-glutamine for increased stability . Add 45 ml of 200 mM L-glutamine per 500 ml medium prior to use .Insect cell culture tested .Recommended storage conditions: 2°C to 8°C, in the dark .

Reference1 . Godwin, G .P ., Danner, D ., and Gorfien, S .F . (1995) FOCUS® 17, 58 .


1,000 ml 10486-025

Related products PageL-Glutamine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .149

Grace’s Insect Cell Culture Medium, supplemented (1X), liquidInsect cell culture tested .Recommended storage conditions: 2°C to 8°C, in the dark .


500 ml 11605-045

10 × 500 ml (case) 11605-086

Contains 3,330 mg/L lactalbumin hydrolysate, 3,330 mg/L yeastolate, and L-glutamine, but no insect hemolymph .

Grace’s Insect Cell Culture Medium, supplemented (2X), liquidInsect cell culture tested .Recommended storage conditions: 2°C to 8°C, in the dark .


100 ml 11667-037

Contains 6,660 mg/L lactalbumin hydrolysate, 6,660 mg/L yeastolate, and L-glutamine .

Grace’s Insect Cell Culture Medium, unsupplemented (1X), liquidInsect cell culture tested .Recommended storage conditions: 2ºC to 8ºC, in the dark .


500 ml 11595-030

Contains L-glutamine, but no L-methionine or insect hemolymph .

IPL-41 Insect Medium (1X), liquidInsect cell culture tested .Recommended storage conditions: 2°C to 8°C, in the dark .


1,000 ml 11405-081

Contains L-glutamine, calcium chloride, and sodium bicarbonate, but no tryptose phosphate broth .


100


Customized Insect Medium ProductsWe will customize the Insect Cell Expressions products formulations to best suit your system’s needs . Please contact our Custom Products Services group for assistance in selecting the products and packaging that best meet your requirements Please note that Sf900™ II Serum-free Medium has been found to be useful in insect cell culture systems other than those employing Sf9 or Sf21 lines . .

Insect Cell Culture Media FormulationsBase cat. no. 11300 11605 11595 11667 11405 21720

ComponentGrace’s*

powder, mg/LGrace’s* supplemented

1X liquid, mg/LGrace’s* unsupplemented

1X liquid, mg/LGrace’s*

2X liquid, mg/LIPL-41L

1X liquid, mg/LSchneider’s† ‡ §

1X liquid

Inorganic salts

CaCl2 750 .00 500 .00 750 .00 1,000 .00 500 .00 794 .00

CuCl2•2H2O – – – 0 .20 0 .20

CoCl2•6H2O – – – – 0 .05 –

FeSO4•7H2O – – – – 0 .55 –

KCl 4,100 .00** 2,800 .00 4,100 .00 5,600 .00 1,200 .00 1,600 .00

KH2PO4 – – – – – 450 .00

MgCl2 1,070 .00 1,070 .00 – 2,138 .64 – –

MgCl•4H2O – – – – 0 .02 –

MgCl2•6H2O – – 2,280 .00 – – –

MgSO4 1,358 .00 1,358 .00 – 2,715 .34 918 .00 3700 .00

MgSO4•7H2O – – 2,780 .00 – – –

MnCl2•4H2O – – – – 0 .02 –

NaCl – – – – 2,850 .00 2,100 .00

NaHCO3 – 350 .00 350 .00 700 .00 350 .00 400 .00

NaH2PO4•H2O 1,013 .00 1,013 .00 1,013 .00 2,026 .00 1,160 .00 –

NaH2PO4•2H2O – – – – – –

NaH2PO4•7H2O – – – – 1321 .00

Na2HPO4 – – – – – 794 .00

(NH4)6(Mo7O24•4H2O) – – – – 0 .04 –

ZnCl2 – – – – 0 .04 –

Other components

D-Fructose 400 .00 400 .00 400 .00 800 .00 – –

Fumaric acid 55 .00 55 .00 55 .00 110 .00 4 .40 100 .00

D-Glucose 700 .00 700 .00 700 .00 1,400 .00 2,500 .00 2,000 .00

α-Ketoglutaric acid 370 .00 370 .00 370 .00 740 .00 29 .60 200 .00

Malic acid 670 .00 670 .00 670 .00 1,340 .00 53 .60 –

Maltose – – – – 1,000 .00 –

Succinic acid 60 .00 60 .00 60 .00 120 .00 4 .80 100 .00

Sucrose 26,680 .00 26,680 .00 26,680 .00 53,360 .00 1,650 .00 –

Trehalose•2H2O – – – – – 2,000 .00

N2 Amine YT – – – – – –

Amino acids

L-Alanine 225 .00 225 .00 225 .00 450 .00 – –

β-Alanine 200 .00 200 .00 200 .00 400 .00 300 .00 500 .00

* Grace, T .D . (1962) Nature 195, 788 . † Schneider, Imogene (1964) Exp. Zool. 156:1, 91 . ‡ Revision of original formulation, Schneider, Imogene (1965) personal communications . § Original formulations list Na2HPO4 at 700 .00 mg/L . Revised formulation believed by author to possess better buffering capacity and better nutritive properties . ** Increased from 2,240 .00 mg/L upon recommendation of Dr . Vaughn .

continued



4



Insect Cell Culture Media Formulations, continuedBase cat. no. 11300 11605 11595 11667 11405 21720

ComponentGrace’s*

powder, mg/LGrace’s* supplemented

1X liquid, mg/LGrace’s* unsupplemented

1X liquid, mg/LGrace’s*

2X liquid, mg/LIPL-41L

1X liquid, mg/LSchneider’s† ‡ §

1X liquid


L-Arginine – – – – – 400 .00

L-Arginine•HCl 700 .00 700 .00 700 .00 1,400 .00 800 .00 –

L-Asparagine 350 .00 350 .00 350 .00 700 .00 1,300 .00 –

L-Aspartic acid 350 .00 350 .00 350 .00 700 .00 1,300 .00 400 .00

L-Cysteine – – – – – 60 .00

L-Cystine – – 22 .00 – – 100 .00

L-Cystine•2HCl 28 .68 28 .68 – 57 .36 – –

L-Cystine•2Na – – – – 119 .14 –

L-Glutamic acid 600 .00 600 .00 600 .00 1200 .00 1,500 .00 800 .00

L-Glutamine 600 .00 600 .00 600 .00 1,200 .00 1,000 .00 1,800 .00

Glycine 650 .00 650 .00 650 .00 1,300 .00 200 .00 250 .00

L-Histidine 2,500 .00 2,500 .00 2,500 .00 5,000 .00 200 .00 400 .00

L-Histidine HCl•H2O – – – – – –

L-Hydroxyproline – – – – 800 .00 –

L-Isoleucine 50 .00 50 .00 50 .00 100 .00 750 .00 150 .00

L-Leucine 75 .00 75 .00 75 .00 150 .00 250 .00 150 .00

L-Lysine•HCl 625 .00 625 .00 625 .00 1,250 .00 700 .00 1,650 .00

L-Methionine 50 .00 50 .00 – 100 .00 1,000 .00 800 .00

L-Phenylalanine 150 .00 150 .00 150 .00 300 .00 1,000 .00 150 .00

L-Proline 350 .00 350 .00 350 .00 700 .00 500 .00 1,700 .00

L-Serine 550 .00 550 .00 550 .00 1,100 .00 200 .00 250 .00

L-Threonine 175 .00 175 .00 175 .00 350 .00 200 .00 350 .00

L-Tryptophan 100 .00 100 .00 100 .00 200 .00 100 .00 100 .00

L-Tyrosine – – 50 .00 – – 500 .00

L-Tyrosine•2Na 62 .14 62 .14 – 100 .00 360 .40 –

L-Valine 100 .00 100 .00 100 .00 200 .00 500 .00 300 .00

Vitamins

Para-aminobenzoic acid 0 .02 0 .02 0 .02 0 .04 0 .32 –

Biotin 0 .01 0 .01 0 .01 0 .02 0 .16 –

D-Calcium pantothenate 0 .02 0 .02 0 .02 0 .04 0 .008 –

Choline chloride 0 .20 0 .20 0 .20 0 .40 20 .00 –

Folic acid 0 .02 0 .02 0 .02 0 .04 0 .08 –

i-Inositol 0 .02 0 .02 0 .02 0 .04 0 .40 –

Niacin 0 .02 0 .02 0 .02 0 .04 0 .16 –

Pyridoxine HCl 0 .02 0 .02 0 .02 0 .04 0 .40 –

Riboflavin 0 .02 0 .02 0 .02 0 .04 0 .08 –

Thiamine HCl 0 .02 0 .02 0 .02 0 .04 0 .08 –

Vitamin B12 – – – – 0 .24 –

Other components

Lactalbumin Hydrolysate – 3,303 .00 – 6,660 .00 – –

Yeastolate – 3,330 .00 – 6,660 .00 – 2,000 .00

* Grace, T .D . (1962) Nature 195, 788 . † Schneider, Imogene (1964) Exp. Zool. 156:1, 91 . ‡ Revision of original formulation, Schneider, Imogene (1965) personal communications . § Original formulations list Na2HPO4 at 700 .00 mg/L . Revised formulation believed by author to possess better buffering capacity and better nutritive properties . ** Increased from 2,240 .00 mg/L upon recommendation of Dr . Vaughn .


102


Insect Medium Supplements

4% Agarose GelRecommended storage conditions: 2°C to 8°C .


40 ml 18300-012

Chemically Defined Lipid ConcentrateChemically Defined Lipid Concentrate is a concentrated lipid emulsion designed to reduce or replace fetal bovine serum in cell culture media for a wide variety of applications, including growth and maintenance of CHO, hybridoma, and insect cells in culture; monoclonal antibody production by hybridomas; and viral expression in insect cells . The components of this medium supplement are chemically defined .For product formulation please refer to the tables on page 162 .

Insect cell culture tested .Recommended storage conditions: 2°C to 8°C, in the dark .Attention: This material is hazardous . All persons using this product should review the Material Safety Data Sheet before handling .


100 ml 11905-031

Pluronic® F-68, 10% (100X)Prepared in distilled water .Insect cell culture tested .Recommended storage conditions: 15°C to 30°C .



100 ml 24040-032

Yeastolate Ultrafiltrate (50X), liquidYeastolate Ultrafiltrate is formulated specifically for use with IPL-41 Insect Medium to support the growth of Spodoptera frugiperda (Sf9) . Prepared with 200 g/L of yeastolate in distilled water .

Insect cell culture tested .Performance testing: Performance tested in a growth assay using Sf9 cells .


100 ml 18200-048

Cellfectin® ReagentCellfectin® II Reagent is a proprietary, cationic lipid formulation in membrane filtered water suitable for DNA transfection into insect cells . Cellfectin ® II Reagent is an improved version of the Cellfectin® Reagent and delivers the same performance as Cellfectin® with a faster protocol . Transfection using Cellfectin® II results in consistent and efficient transfection of Sf9, Sf21, and High Five™ cells in Bac-to Bac®, BaculoDirect™, and InsectSelect™ Expression Systems from Invitrogen or equivalent systems . Cellfectin® Reagent can also be used to transfect adherent or suspension mammalian cells .Transfection protocols for Sf9 and Sf21 cells are described in this manual . For transfecting High Five™ insect cells and mamamalian cells with Cellfectin® II, refer to the transfection selection tool at www .invitrogen .com/transfection Important Guidelines for Transfection

Cellfectin® II Reagent is a lipid suspension that may settle with time . ■

Mix thoroughly by inverting the tube 5 – 10 times before use .The DNA:lipid complex formation time is shorter (~15-30 minutes) ■

when using Cellfectin® as compared to Cellfectin® ReagentPerform transfection using the appropriate protocol for the system ■

and cell type that you are using to obtain the best results .Do not add antibiotics to media during transfection as this will cause ■

cell death .Transfection complexes must be formed in serum-free medium ■

Test seum-free medium for compatibility with Cellfectin® II since some ■

serum-free formulations may inhibit lipid-mediation transfectionOptimizing TransfectionsTo optimize the highest transfection efficiency and minimize non-specific effects, optimize transfection conditions by varying cell density, DNA and Cellfectin® concentrations, and transfection incubation time .


1 ml 10362-100

125 μl 10362-125



4



Transfecting Sf9 or Sf21 with Baculovirus Use the following procedure to transfect Sf9 or Sf21 insect cells cultured in Grace’s Insect Medium, supplemented, containing 10% FBS in a 6-well format .All amounts and volumes are given on a per well basis . 1 . If the cell density is in the range o 1 .5-2 .5 x 106 cells ml with 95% viability, and

the medium is without antibiotics, proceed to step 1a . If the cell density is not in this range or the medium contains antibiotics, follow steps 1b-1c . a . Add 2ml of Grace’s Insect Medium, Unsupplemented (without

antibiotics and serum) in each well . Plate 8 x 105 Sf9 or Sf21 cells per well . Do not change medium or wash the cells . The medium carried over to the next step will enhance the transfection efficiency . Allow cells to attach for 15 minutes at room temperature in the hood . Proceed to step 2 .

b . Prepare 10ml plating medum by mixing 1 .5ml Grace’s Insect Medium, Supplemented, containing 10%FBS (without antibiotics) and 8 .5ml Grace’s Insect Medium, Unsupplemented (without FBS and antibiotics) .

c . Plate 8 x 105 Sf9 or Sf21 cells per well . Allow cells to attach for 15mins at room temperature . DNA in the hood . Remove the medium . Add 2 .5ml plating medium from step 1b per well . Proceed to step 2 .

2 . For each transfection sample , prepare complexes as follows: Bac-to bac® System

a . Mix Cellfectin ® before use and dilute 8 µl in 100 ul Grace’s Insect Medium, Unsupplemented . Vortex briefly to mix ‘

Note: This mixture can be kept at room temperature for up to 30 minutes .

b . Dilute 1ug Grace’s Insect Medium, Unsupplemented . Mix Gently .

c . Combine the diluted DNA with diluted Cellfectin ® II (total volume is ~ 210µl) . Mix gently and incubate for 15 – 30 minutes at room temperature .

Baculo™ Direct System a . Prepare Transfection Mix A: Cellfectin® II 8µl Grace’s Insect Medium 100ul UnsupplementedNote: This mixture can be kept at room temperature for up to 30 minutes .

b . Prepare Transfection Mix B: LR recombination reaction 10µl Grace’s Insect Medium 100µl Unsupplemented

c . Combine Transfection mixtures A and B . Mix gently and incubate for 25 – 35 minutes at room temperature .

3 . Add-210ul DNA mixture or transfection mixture (Step 2c) dropwise onto the cells from Step 1a or 1c . Incubate cells at 27oC for 3-5 hours .

4 . Remove the transfection mixture and replace with 2ml of complete growth medium(e .g . Grace’s Insect Medium, Supplemented and 10% FBS) . Use of antibiotics is optional for bac-to bac® system . Use complete medium with 100uM ganciclovir for Baculodirect™ system .

5 . Incubate cells at 270C for 72 hours or until you see signs of viral infection .


104


Transfecting Sf9 or Sf21 with Plasmid DNA Use this procedure to transfect Sf9 and Sf21 Insect cells in a 6-well format in Sf900 III SFM, Sf-900™, or Grace’s Insect Medium . All amounts and volumes are given on a per well basis .1 . For log phase cells cultured in Sf-900 II SFM or Sf-900™ III SFM

without antibiotics: Plate 8 x 105 Sf9 or Sf21 cells per well in 2ml Sf-900 II SFM or Sf-900 ™ III SFM . Allow cells to attach for 15 minutes at room temperature . Proceed to Step 2 .

For cells cultured in of Grace’s Insect Medium: If the cell density is not in this range or the medium contains antibiotics, follow steps 1b – 1c .

a . Add 2ml of Grace’s Insect Medium, Unsupplemented (without antibiotics and serum) in each well . Plate 8 x 105 Sf9 or Sf21 cells per well . Do not change medium or wash the cells . The medium carried over to the next step will enhance the transfection efficiency . Allow cells to attach for 15 minutes at room temperature in the hood . Proceed to Step 2 .

b . Prepare 10ml plating medium by mixing 1 .5ml Grace’s Insect Medium, Supplemented, containing 10% FBS (without antibiotics) and 8 .5ml Grace’s Insect Medium, Unsupplemented, (without FBS and antibiotics) .

c . Plate 8 x 105 Sf9 or Sf21 cells per well . Allow cellsnto attach for 15 minutes at room temperature in the hood . Remove the medium . Add 2 .5ml plating medium from step 1b per well, Proceed to step 2 .

2 . For each transfection sample, prepare complexes as follows: a . Dilute 1 µg plasmid DNA in 100 µl Grace’s Insect Medium,

Unsupplemented (without antibiotics and serum) . Vortex briefly to mix, Incubate at room temperature for 15-30 minutes .

Optional: If you are using SFM, add 5 µl PLUS™ Reagent (Cat .no . 11514-015) to the diluted DNA to improve transfection efficiency .

b . Mix Cellfectin™ II before use, then dilute 8 µl Cellfectin™ II in 100µl Grace’s Insect Medium, unsupplemented (without antibiotics and serum) . Vortex briefly to mix . Incubate at room temperature for 15 – 30 minutes.

c . Combine the diluted DNA diluted Cellfectin® II (total volume is ~210 µl) . Mix gently and incubate for 15 – 30 mins .

3 . Add ~ 210 µl DNA lipid mixture (Step 2c) dropwise onto the cells from Step 1a or 1c .

4. For Sf-900 II SFM or Sf-900 ™ III SFM: Incubate cells at 27oC for 24–48 hours and test for transgene expression . There is no need to replace the medium .

5. For Grace’s medium: Incubate cells at 27oC for 3-5 hours . Remove the transfection mixture and replace with 2ml of complete growth medium (e .g . Grace’s, Supplemented, and 10% FBS)> Addition of antibiotics to the growth medium is optional . Incubate cells at 27oC for 24–48 hours and test for transgene expression .

Quality ControlThe Certificate of Analysis (CofA) Provides detailed quality control information for each product . Certificates of analysis are available from www .invitrogen .com/support and search for the Certificate of Analysis by product lot number, which is printed on the box

Limited Use Label No.326: Cellfectin® II Transfection ReagentThis product is subject to U .S Patents 4, 897, 355 and 5, 550, 289 . This Product s licensed for use in in vitro delivery of proteins, peptides, or other macromolecules only . For a license to use the product in any other field-of-use, including in vitro uses, contact Roche Palo Alto LLC>< 3431 Hillview Avenue, Palo Alto CA 04304-1397© 2008 Invitrogen Corporation . All rights reserved .For research use only . Not intended for any animal or human therapeutic or diagnostic use .



4



Hybridoma and Monoclonal Antibody Products


FREE CD Hybridoma Medium (1X), liquidCD Hybridoma Medium is a chemically defined, protein-free medium optimized for the growth of a variety of hybridomas and myelomas and the production of monoclonal antibodies in stationary or agitated suspension systems .1,2 CD Hybridoma Medium contains no proteins of animal, plant, or synthetic origin . There are also no undefined lysates or hydrolysates in the formulation .

Formulated without L-glutamine for increased stability; add 40 ml of 200 mM L-glutamine or 40 ml of GlutaMAX™-I Supplement per 1,000 ml of medium prior to use .

CD Hybridoma Medium is not optimized for lipid-dependent or cholesterol-dependent cultures such as NS0-derived lines . Please inquire about options for those applications . Recommended storage conditions: 2°C to 8°C, in the dark .Notes: A Hybridoma Medium Master file has been submitted to the FDA . Permission to cross reference the Master file may be obtained by contacting Invitrogen Technical Support or you local Sales Representative .

References1 . Gorfien, S .F ., Godwin, G .P ., Samrock, R ., and Tilkins, M .L . (1999) FOCUS® 21, 30 .2 . Csirke, B ., Godwin, G .P ., and Gorfien, S .F . (1999) FOCUS® 21, 33 .


1,000 ml 11279-023

Contains no L-glutamine or phenol red .

Related products PageL-Glutamine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .149250X Cholesterol Lipid Concentrate

for growing NS0 and other lipid dependent cell lines . . . . . . . . . . . . .106Hybridoma reagents . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .166


FREE CD Hybridoma AGT™CD Hybridoma AGT™ is a chemically defined, protein-free medium in a new easy-to-use granular format . CD Hybridoma AGT™ is optimized for the growth of a variety of hybridoma and myelomas and the production of monoclonal antibodies in stationary or agitated suspension systems .Recommended storage conditions: 2°C to 8°C, in the dark .


1 × 1 L 12372-025

1 × 10 L 12372-017

Related Products PageL-Glutamine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .140250X Cholesterol Lipid Concentrate

for growing NS0 and other lipid dependent cell lines . . . . . . . . . . . . . . 30Hybridoma reagents . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .159Distilled Water . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .152

Hybridoma-SFM (1X), liquidHybridoma-SFM is a very low–protein (containing 20 µg/ml protein as insulin and transferrin) medium optimized for the growth of a variety of hybridomas and the production of monoclonal antibodies . Most hybrid omas require only minor sequential adaptation or no adaptation to this medium . The very low protein content facilitates monoclonal antibody purification .Recommended storage conditions: 2°C to 8°C, in the dark .Notes: A Hybridoma Medium Master File has been submitted to the FDA . Permission to cross-reference the Master File may be obtained by contacting Invitrogen Technical Support or your local Sales Representative .

Reference1 . Mullins, T .D ., Dzimian, J .L ., Fike, R .M ., and Weiss, S .A . (1991) FOCUS® 13, 91 .


500 ml 12045-084

1,000 ml 12045-076


Related products PageHybridoma reagents . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .166

250X Cholesterol Lipid Concentrate250X Cholesterol Lipid Concentrate is a chemically defined, protein-free supplement used for growing NS0 and other lipid-dependent cell lines to high densities while supporting growth and protein expression without complicating downstream purification . The supplement can be added to 1X media such as CD Hybridoma and filtered with minimal loss of lipids . Custom formulations and packaging configurations as well as supplements used in feeding strategies are available through our Custom Products Services group .Recommended storage conditions: 2°C to 8°C, in the dark .

Reference1 . Gorfien, S ., Paul, W ., Walowitz, J ., Keem, R ., Biddle, W . and Jayme, D . (2000),

Biotechnol. Prog. 16, 682-687 .


100 ml 12531-018


106



FREE PFHM-II Protein-free Hybridoma Medium (1X), liquidPFHM-II is a protein-free, ready-to-use medium that contains no poly pep tide growth or attachment factors, or mediators that may com-plicate downstream processing and final product purification .1 PFHM-II performs as well as serum-supplemented media for MAb production and also may be used as a growth medium .

In instances where antibiotics are required, most general antibiot-ics are compatible with PFHM-II, including penicillin/streptomycin, genta micin, anti-PPLO, linocin, and Fungizone™ Reagent . Do not use kanamy cin sulfates or neomycin sulfates .Recommended storage conditions: 2°C to 8°C, in the dark .

Fungizone™ is a trademark of E .R . Squibb & Sons, L .L .C .

Reference1 . Fike, R ., Pfohl, J ., Jayme, D ., and Weiss, S .A . (1990) FOCUS® 12, 79 .


1,000 ml 12040-077


Medium NCTC-109 (1X), liquidNCTC-109 (1X) is a synthetic medium that is suitable for general use in generating and maintaining hybridoma cells . This medium typically requires the addition of a growth factor cocktail or serum .Recommended storage conditions: 2°C to 8°C .Intended use: In vitro diagnostic (IVD) .


500 ml 21340-054

Contains no L-glutamine .

Customized Hybridoma Medium ProductsHybridoma and Monoclonal Antibody products, along with DMEM/F-12, IMDM/F-12, and your in-house formulations are available on a make-to-order basis . We realize that not all needs are alike, so we’ve developed the capabilities to provide the desired configuration (formulation and packaging) of product type, as well as the operational systems that can provide these products in a timely fashion . Please contact our Custom Products Services group for assistance in selecting the products and packaging that best meet your requirements .

Additional considerations for optimizing hybridoma systems include the use of nutrient supplements (specially developed for your system or general) and glutamine dipeptides (see GlutaMAX™ Supplement, page 54) for enhanced stability .

HAT Supplement (50X), liquidHAT Supplement (50X) is a liquid mixture of sodium hypoxanthine, ami nop terin, and thymidine . After dilution, HAT is suitable for use as a post-fusion selective medium to eliminate unfused or self-fused HGPRT-myeloma cells . Hypoxanthine and thymidine supply preformed purines and pyrimidines for DNA synthesis by hybridomas via the salvage pathway that utilizes HGPRT contributed by the fused spleen cell . Ami nopterin, a folic acid antagonist, inhibits the de novo nucleoside bio syn thesis pathway .Concentration at 50X: 5 mM sodium hypoxanthine, 20 µM aminopterin, 0 .8 mM thymidine .Recommended storage conditions: –5°C to –20°C .


100 ml 21060-017

HT Supplement (100X), liquidHT Supplement (100X) liquid is a mixture of sodium hypoxanthine and thymidine . After dilution, HT Supplement is suitable for use as a rescue medium . The supplement provides preformed purines and pyrimidines to overcome the effects of residual intracellular aminopterin . Once the de novo biosynthesis pathway for nucleosides has been re-established, HT supplementation can be discontinued .Concentration at 100X: 10 mM sodium hypoxanthine, 1 .6 mM thymidine .Recommended storage conditions: 2°C to 8°C .


50 ml 11067-030



4



Protein A AgaroseProtein A Agarose is suitable for purifying IgG molecules from a variety of species . Protein A Agarose also can be used for fractionation of IgG fragments, fractionation of cells tagged with IgG, and isolation of antigen-antibody complexes . In addition, Protein A interacts with some classes of IgM and IgA . The matrix consists of purified Protein A covalently attached to cross-linked 4% agarose beads using the cyanogen bromide method . Protein A Agarose can bind >18 mg of human IgG per ml of gel .Performance and quality testing: Binding capacity for human IgG is determined by affinity chromatography .Concentration in storage buffer: 45% to 55% suspension in 20 mM sodium phosphate (pH 7 .2), 0 .15 M NaCl, 0 .02% (w/v) merthiolate .Recommended storage conditions: 4°C .

Protein A Agarose is manufactured under license from Amersham Pharmacia Biotech, Inc .


5 ml 15918-014

Recombinant Protein G (rProtein G) AgaroseRecombinant Protein G (rProtein G) Agarose is suitable for purification of IgG antibodies using column chromatography or immunoprecipitation methods . This matrix, which contains a genetically engineered form of streptococcal Protein G, is designed to eliminate binding of serum albumin while maintaining efficient binding of the Fc region of IgG isotypes . rProtein G is produced in E. coli and, after purification, is covalently immobilized on 4% agarose beads by cyanogen bromide . rProtein G Agarose binds to a broader range of IgG isotypes than react with Protein A . rProtein G Agarose can bind >18 mg of human IgG per ml of gel . The binding characteristics and stability of rProtein G Agarose facilitate rapid and repeated use for affinity chromatography .

rProtein G Agarose is supplied as a 50% suspension in PBS (pH 7 .0), 20% ethanol . Recommended storage conditions: 4°C .


5 ml 15920-010

Streptavidin AgaroseStreptavidin Agarose is suitable for isolating biotinylated molecules and complexes with biotinylated components, including antibod-ies, antibody-antigen complexes, ligands and receptors, nucleic acids, and nucleic acid-protein complexes . It is especially useful as an immunoprecipitation reagent for antibodies that do not bind well to Protein A . The matrix consists of streptavidin covalently attached to 4% agarose beads through a 15-atom spacer arm . Streptavidin Agarose can bind 3 to 8 mg of biotinylated IgG per ml of gel .

Streptavidin Agarose is supplied as 10 ml of a 50% slurry containing 5 ml of packed gel bed in 0 .01 M sodium phosphate (pH 7 .2), 0 .15 M NaCl, and 0 .02% sodium azide . Recommended storage conditions: 4°C .


5 ml SA100-04


108


Cellular StudiesBronchial Epithelial Products

LHC Medium (1X), liquidFormulated for the growth of bronchial epithelial cells with further supplements .

LHC serum-free media are the most widely used products for culture of bronchial epithelial cells . For maximum flexibility and convenience, we offer these products with or without supplementation .Recommended storage conditions: 2°C to 8°C .


LHC Basal Medium (1X), liquid 500 ml 12677-019 1,000 ml 12677-027

LHC-8 Medium (1X), liquid 500 ml 12678-017

Recommended applications

Complete media (for cell culture for the following research/disease areas: asthma, allergies, lung cancer, cystic fibrosis, pulmonary and esophageal cancer*)

Typical cells cultured

Media supplements

LHC productRetinoic acid Epinephrine GentamicinOther

supplements †

IB3-1, S9, C38 ● ● LHC-8

BEAS-2B, BZR, BBM, Het-1A, NHBE BBE

●LHC-8 w/o gentamicin

● ● ● ● LHC-9

Basal media (for flexible customization) LHC Basal

* For research use only . † Includes insulin, hydrocortisone, EGF, transferrin, bovine pituitary extract, and T3 .



4



Dermatology Products

Defined Keratinocyte-SFM (1X), liquidDefined Keratinocyte-SFM is a serum-free medium optimized for the isolation and expansion of human keratinocytes without the need for bovine pituitary extract (BPE) supplementation or the use of fibroblast feeder layers .1,2 The growth-promoting activity of BPE has been replaced by the inclusion of growth promoting agents which enhance consistent product performance . These growth-promoting agents also maintain cell morphology, physiological markers, and growth . The medium is formulated to inhibit fibroblast proliferation and has a low calcium concentration (<0 .1 mM) allowing for the isolation and culture of highly enriched populations of undifferentiated keratinocytes .Recommended storage conditions: medium, 2°C to 8°C, in the dark; supplement, –5°C to –20°C .Shelf life after supplementation: 10 weeks .Notes: This formulation contains some human and animal-origin components .

A Defined Keratinocyte-SFM Master File has been submitted to the FDA . Permission to cross-reference the Master File may be obtained by contacting Invitrogen nical Support or your local Sales Representative .

Defined Keratinocyte-SFM consists of a basal liquid medium and frozen supplement which is added at the time of use . The fully supplemented medium has a shelf life of 10 weeks under normal use conditions . Product performance is confirmed by evaluation of human primary keratinocyte growth .

References1 . Judd, D .A ., Battista, P .J . and Behm, D .D . (1997) FOCUS® 19, 2 .2 . Donovan, J . (1998) FOCUS® 20, 38 .


500 ml 10744-019

Related products PageCell Dissociation Products . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .158Primary Human Keratinocytes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .179

Keratinocyte Starter KitThe Keratinocyte Starter Kit includes Defined Keratinocyte-SFM Medium, growth supplement, and Primary Human Keratinocytes .Recommended storage conditions: medium, 2°C to 8°C, in the dark; supplement, –5°C to –20°C; cells, –125°C to –200°C (liquid nitrogen) .


1 kit 12568-010

Keratinocyte-SFM (1X), liquidKeratinocyte-SFM is a serum-free medium (with BPE) optimized for the cultivation of primary and passaged human keratinocytes .1,2 Keratino cyte-SFM can be used with or without an irradiated fibroblast feeder layer . The formulation retards fibroblast overgrowth, thereby allowing the study of highly pure populations of keratinocytes . The low calcium ion concentration (<0 .1 mM) permits greater cell growth and slows cell differentiation .

Keratinocyte-SFM is supplied with prequalified human recombinant Epidermal Growth Factor 1-53 (EGF 1-53), and Bovine Pituitary Extract (BPE) . Separate packaging of these components allows flexibility in determining the supplement concentrations most suited to the application .Recommended storage conditions: medium, 2°C to 8°C, in the dark; supplements: –5°C to –20°C .


500 ml 17005-042


References1 . Daley, J .P ., Epstein, D .A ., and Hawley-Nelson, P . (1990) FOCUS® 12, 68 .2 . Donovan, J . (1998) FOCUS® 20, 38 .

Keratinocyte-SFM (1X), liquid (without CaCl2)Keratinocyte-SFM is supplied with prequalified human recombinant Epidermal Growth Factor 1-53 (EGF 1-53), and Bovine Pituitary Extract (BPE) in separate packaging . Components are not sold separately .Recommended storage conditions: medium, 2°C to 8°C, in the dark; supplements: –5°C to –20°C .Shelf life after supplementation: 2 weeks .


500 ml 37010-022

Contains L-glutamine but no calcium chloride .

Hematology-Oncology Research Products (AIM V® Media)Serum-free media developed for research applications using various differentiated cell types may also be evaluated for biomedical research applications . Although products and components are screened for absence of contaminants, we recommend that users exercise caution in handling all products containing materials of human/animal origin and encourage handling practices consistent with materials that are potentially infectious . It is the exclusive responsibility of the end user to qualify/validate these products for specific applications .


110


Cell Types Successfully Cultivated Using Serum-free AIM V® MediumCell line/type Application Media used previously*

Human primary lymphoid cells

T cells Activation RPMI 1640

T cells Cloning RPMI 1640 †

T cells Retrovirus research RPMI 1640

CD8+ T lymphocytes CTL induction RPMI 1640

Macrophages Differentiation RPMI 1640, IMDM

Human monocytes Growth RPMI 1640

Transformed lymphoid lines

HUT78 Retrovirus research RPMI 1640, IMDM §

HL-60 Nuclear transcription research DMEM

COS cells (monkey) Protein secretion DMEM

YAC-1 (mouse) IL-2 activation RPMI 1640 ‡

Murine lymphoma Growth DMEM

Myeloid cells Growth RPMI 1640

Hybridoma Monoclonal antibody production RPMI 1640, IMDM

Leukemic cells Immune regulation RPMI 1640 ‡

Adherent cells

Oral epithelial Growth DMEM

Human fibroblasts Growth RPMI 1640

VERO Growth MEM

* Supplemented with 10% fetal bovine serum unless otherwise indicated . † Supplemented with 10% human serum . § Supplemented with 20% newborn calf serum . ‡ Supplemented with 10% newborn calf serum .

AIM V® Medium for Lymphocyte CultureAIM V® Medium (serum-free lymphocyte medium) was designed specifically for research purposes in adoptive immunotherapy, a method of cancer treatment explored in clinical trials . The medium has, however, been found to be useful in a growing list of applications, including culturing functionally differentiated lymphoid cell lines, investigating signal transduction pathways, and performing applications requiring component definition . The medium has applications in the following areas: lymphokine-activated killer (LAK) cell induction and function,1–7 cytokine and antibody activity in LAK cell induction,8–12 long-term cultivation of functional human macrophages,13 immunotherapy of murine hepatic metastases,14 lymphocyte receptor studies,15 and ADCC-dependent lymphocyte activation .16

As shown in the table, AIM V® Medium has been used for serum-free growth of many cell types . The medium may also have applications in the following areas of research: NK cells and NK-like effector populations; T cell activation; cytokine regulation of effector cells; cytokine interactions and cascades; cytokine receptor and post receptor events in lymphoid effector cells; macrophage activation; effector cells in anti tumor immunity; mechanisms of cell-mediated cytotoxicity; in vitro effects of lymphokines, antibodies, and immunotherapy; hybridoma growth and monoclonal antibody production; AIDS research; production of HIV proteins (e .g ., gP160 and gP120); and definition and isolation of regulatory proteins produced by activated monocytes and macrophages .

References1 . Muul, L .M ., Nason-Burchenal, K ., Hyatt, C .,

Schwarz, S ., Slavin, D ., Director, E .P ., and Rosen-berg, S .A . (1987) J. Immunol. Methods 105, 183 .

2 . Jadus, M .R ., Thurman, G .B ., Mrowca-Bastin, A ., and Yanelli, J . (1988) J. Immunol. Methods 109, 169 .

3 . Grimm, E .A ., Owen-Schaub, L .B ., Loudon, W .G ., and Yagita, M . (1988) Ann. N.Y. Acad. Sci. 532, 788 .

4 . Beckner, S .K ., Maluish, A .E ., and Longo, D .L . (1987) Cancer Res. 47, 5504 .

5 . Ryan, T ., Atkins, M .B ., Mier, J .W ., and Beckman, E .M . (1989) Transfusion 29, 491 .

6 . Knazek, R .A ., Ulu, Y-W ., Aebersold, P .M ., and Rosenberg, S .A . (1990) J. Immunol. Methods 127, 29 .

7 . Haas, G .P ., Solomon, D ., and Rosenberg, S .A . (1990) Cancer Immunol. Immunother. 30, 342 .

8 . Owen-Schaub, L .B ., Gutterman, J .V ., and Grimm, E .A . (1988) Cancer Immunol. Immunother. 29, 193 .

9 . Yang, S .C ., Owen-Schaub, L .B ., Grimm, E .A ., and Roth, J .A . (1989) Cancer Immunol. Immunother. 29, 193 .

10 . Grimm, E .A ., Crump III, W .L ., Durett, A ., Hester, J .P ., Lagoo-Deenadalayan, S ., and Owen-Schaub, L .B . (1988) Cancer Immunol. Immunother. 27, 53 .

11 . Crump III, W .L ., Owen-Schaub, L .B ., and Grimm, E .A . (1989) Cancer Res. 49, 149 .

12 . Yang, S .C ., Owen-Schaub, L .B ., Grimm, E .A ., and Roth, J .A . (1988) Surg. Forum 39, 454 .

13 . Helinski, E .M ., Bielat, K .L ., Ovak, G .M ., and Pauly, J .L . (1988) J. Leukocyte Biol. 44, 111 .

14 . Lafreniere, R ., Borkenhagen, K ., Bryant, L .D ., and Huchcroft, S . (1989) Cancer Res. 49, 2409 .

15 . Owen-Schaub, L .B ., Crump III, W .L ., Morin, G .I ., and Grimm, E .A . (1989) J. Immunol. 143, 2336 .

16 . Masacci, G ., Weroall, P ., Nielsen, J ., Kierulff-Nielsen, H ., Wizzell, H ., and Mellstedt, M . (1989) Hybridoma 8, 507 .



4



AIM V® Medium, liquid (research grade)Performance testing: Performance tested in a growth assay using Jurkat cells (ATCC Jurkat T1B-152) .Viral testing: Raw components of human origin are nonreactive, at the donor level, for Hepatitis B surface antigen (HBsAG), antibody to Hepa titis C virus (anti-HCV), antibodies to Human Immunodeficiency Viruses 1 and 2 (anti-HIV 1 and 2), and syphilis, as determined by currently approved testing procedures . Raw material components of human origin are also tested for alanine aminotransferase (ALT) enzyme, and found to be within normal limits . Where possible, such components are further processed by heat treatment in liquid form at 60°C for 10 hours by validated methods demonstrated to inactivate adventitious viral contam inants (performed by our raw material vendors) .Recommended storage conditions: 2°C to 8°C, in the dark .


AIM V® Medium, liquid (research grade, with HSA) 500 ml 12055-091 1,000 ml 12055-083

Contains L-glutamine, streptomycin sulfate at 50 µg/ml, and gentamicin sulfate at 10 µg/ml .


AIM V® Medium, liquid (research grade, with BSA) 500 ml 31035-025

Contains AlbuMAX® supplement (bovine serum albumin) substituted for human serum albumin, L-glutamine, streptomycin sulfate at 50 µg/ml, and gentamicin sulfate at 10 µg/ml .

AIM V® Medium, liquid (therapeutic grade)Performance testing: Performance tested in a growth assay using Jurkat cells (ATCC Jurkat T1B-152) .Viral testing: Raw material components of human origin are non reactive, at the donor level, for Hepatitis B surface antigen (HBsAG), antibody to Hepatitis C virus (anti-HCV), antibodies to Human Immunodeficiency Viruses 1 and 2 (anti-HIV 1 and 2), and syphilis, as determined by currently approved testing procedures . Raw material components of human origin are also tested for alanine aminotrans ferase (ALT) enzyme, and found to be within normal limits . Where possible, such components are further processed by heat treatment in liquid form at 60°C for 10 hours by validated methods demonstrated to inactivate adventitious viral contaminants (performed by our raw material vendors) .Intended uses: For human ex vivo tissue and cell culture processing applications .Recommended storage conditions: 2°C to 8°C, in the dark .Intended uses: For human ex vivo tissue and cell culture processing applications .Notes: An AIM V® Medium Master File has been submitted to the FDA . Permission to cross-reference the Master File may be obtained by contacting Invitrogen Technical Support or your local Sales Representative .


1,000 ml 087-0112DK

10 L 087-0112BK

Contains L-glutamine, 50 µg/ml streptomycin sulfate, and 10 µg/ml gentamicin sulfate .


112


Hematopoiesis Products

StemPro®-34 SFM (1X), liquidStemPro®-34 SFM is a serum-free medium specifically formulated to support the development of human hematopoietic cells in culture .1 StemPro®-34 SFM can be used to study hematopoietic cells from bone marrow, peripheral blood, or neonatal cord blood . StemPro®-34 SFM is performance-tested on normal human bone marrow . It is manufactured with components of human, recombinant, or synthetic origin .StemPro®-34 SFM consists of a basal liquid medium and frozen supplement (StemPro®-Nutrient Supplement) which is added at the time of use .Recommended storage conditions: medium, 2°C to 8°C; supplement, –5°C to –20°C .Notes: An intended use disclosure is required for purchase of StemPro®-34 SFM . For further regulatory information on the uses of this product, please contact the Tech-LineSM .

References1 . Daley, J .P ., Dadey, B .M ., Wysocki, M .G ., Caligiuri, M .A ., and Biddle, W .C . (1996)

FOCUS® 18, 62 . 2 . Taylor, M . et al . (2001) Blood 98, 1195-1199 .3 . Szilvassy, S . et al . (1999) Blood 93, 1557-1566 .4 . Delehanty, L . et al . (2003) Blood 101, 1744-1751 .5 . Kirshenbaum, A ., et al . (1999) Blood 94, 2333-2342 .6 . Szilvassy, S ., Meyerrose, T and Grimes, B . (2000) Blood 95, 2829-2837 .7 . Yahata, T ., et al . (2003) Blood 101, 2905-2913 .8 . Wang, J ., et al . (2003) Blood 101, 2924-2931 .9 . Kolbus, A ., et al . (2003) Blood 102, 3136-3146 .10 . Goyama, S ., et al . (2004) Blood 104, 3558-3564 .


500 ml 10639-011

Macrophage-SFM (1X), liquidMacrophage-SFM is a serum-free medium designed for the culture of human peripheral blood monocytes and macrophages .1


Reference1 . Epstein, D ., Wysocki, M ., Biddle, W ., Streck, R .J ., Rolf, M .M ., and Pauly, J .L . (1991)

FOCUS® 13, 120 .


500 ml 12065-074

Contains L-glutamine, but no antibiotics .

Hepatocyte ProductsGibco® hepatocyte products include a complete range of serum-free media and reagents designed for the isolation and maintenance of hepatocytes, while sustaining the normal pheno typic expression including xenobiotic metabolism .1

Reference1 . Alfonso, J .L ., Samrock, R ., and Price, P . (1995) FOCUS® 17, 6 .

Hepatozyme-SFM (1X), liquidHepatozyme-SFM is a serum-free medium for the long-term main tenance of hepatocyte phenotypic expression, including active and inducible forms of cyto chromes P450 and active phase II enzymes . Recommended storage conditions: 2°C to 8°C, in the dark .


500 ml 17705-021

Contains no phenol red .

Liver Digest Medium (1X), liquidLiver Digest Medium is a collagenase-dispase medium for dissociation of viable liver cells .Recommended storage conditions: –5°C to –20°C, in the dark .


500 ml 17703-034

Liver Perfusion Medium (1X), liquidLiver Perfusion Medium is a buffered, balanced salt solution formulated to cleanse the liver of blood, prevent clotting, and initiate loosening of cell-to-cell contact .Recommended storage conditions: 2°C to 8°C, in the dark .


500 ml 17701-038

Hepatocyte Wash MediumHepatocyte Wash Medium is an enriched Williams’ Medium E for the reduction of cell membrane leakage, ease of centrifugation, and high viability .Recommended storage conditions: 2°C to 8°C, in the dark .


500 ml 17704-024



4



Human Mammary Epithelial Products

HuMEC MediumOptimized specifically for culture of human mammary epithelial cells, HuMEC Medium is a serum-free formulation with frozen supplements containing growth factors and other components . This translates to greater experimental validity, more reproducible results, and reduction of lot-to-lot variability and aberrations that can occur with the addition of serum . Human mammary epithelial cells cultured in HuMEC Medium have gene expression profiles consistent with normal mammary epithelial cells, as tested on a cDNA microarray .

HuMEC Ready Medium includes HuMEC Basal Serum-free Medium and a HuMEC Supplement Kit . The HuMEC Supplement Kit includes 5 ml of a supplement mix containing epidermal growth factor, hydrocortisone, isoproterenol, transferrin, and insulin, and 25 mg of bovine pituitary extract . Applications: HuMEC Medium has been performance tested in a growth assay using normal human mammary epithelial cells . HuMEC Medium allows the culture of human mammary epithelial cells under conditions optimized for breast cancer research, providing researchers in drug discovery and other fields greater insight into carcinogenic progression . Recommended storage conditions: basal medium, 2°C to 8°C; supple-ment mix, –5°C to –20°C .


HuMEC Basal Serum-free Medium (1X), liquid 500 ml 12753-018

HuMEC Ready Medium (1X), liquid 500 ml 12752-010

HuMEC Supplement Kit 1 kit 12755-013

Neurobiology Products

Neurobasal™ MediaNeurobasal™ Media are basal media formulated to specifically meet neuronal cells’ special requirements . They allow for long-term maintenance of the normal phenotype and growth of neuronal cells, and maintain pure populations of neuronal cells without the need for an astrocyte feeder layer .

Neurobasal™ Medium is for long-term growth of fetal hippocampal neurons and many other neurons of the CNS .1

Neurobasal™-A Medium is for long-term growth and viability of rat postnatal and adult hippocampal and cortical neurons of the CNS .2

Prior to use, Neurobasal™ Media must be supplemented with either a serum-free supplement (such as B-27 or N-2) or serum, and 0 .5 mM L-gluta mine or GlutaMAX™-I Supplement . For initial plating, add 25 µM glutamic acid .Recommended storage conditions: 2°C to 8°C, in the dark .

References1 . Brewer, G .J ., Torricelli, J ., Evege, E .K ., and Price, P . (1994) FOCUS® 16, 6 .2 . Brewer, G .J . (1997) J. Neurosci. Methods 71, 145 .3 . Brewer, G ., Price, P . (2001) Protocols for Neural Cell Culture, 3rd Ed ., Ed .: S . Federoff

and A . Richardson, Humana Press, 255–264 .4 . Hermann, A . et al . (2004) J Cell Science 117, 4411–4422 .


Neurobasal™ Medium (1X), liquid 500 ml 21103-049

Neurobasal™ Medium without phenol red (1X), liquid 500 ml 12348-017

Neurobasal™-A Medium (1X), liquid 500 ml 10888-022

Neurobasal™-A Medium without phenol red (1X), liquid 500 ml 12349-015

Contains no L-glutamine, L-glutamic acid, or aspartic acid .

Related products PageL-Glutamine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .149GlutaMAX™-I Supplement . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .123Cell Dissociation Products . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .158TrypLE™ Cell Dissociation Products . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .159Lipofectamine™ 2000 Transfection Reagent . . . . . . . . . . . . . . . . . . . . . . . . 132


114


B-27 Serum-free SupplementsB-27 Supplements are 50X liquid, serum-free supplements for growth and long-term viability of hippocampal neurons .1,2 These supplements are used in combination with a basal medium such as Neurobasal™ Medium .2

B-27 Supplement Minus AO is the complete B-27 Supplement without 5 antioxidants (vitamin E, vitamin E acetate, superoxide dismutase, catalase, and glutathione) that would interfere with studying free-radical damage to neurons .

B-27 Supplement Minus Vitamin A is the complete B-27 Supplement without Vitamin A . The deletion of vitamin A in this supplement prevents neural differentiation of stem cells .Recommended storage conditions: –5°C to –20°C .

References1 . Brewer, G .J ., Torricelli, J .R ., Evege, E .K ., and Price, P .J . (1993) J. Neurosci. Res. 35,

567 .2 . Brewer, G .J ., Torricelli, J .R ., Evege, E .K ., and Price, P .J . (1994) FOCUS® 16, 6 .3 . Brewer, G ., Price, P . (2001) Protocols for Neural Cell Culture, 3rd Ed ., Ed .: S . Federoff

and A . Richardson, Humana Press, 255–264 .4 . Hermann, A . et al . (2004) J Cell Science 117, 4411–4422 .5 . Eide, L . and McMurray, C . (2005) BioTechniques 38, 99–104 .


B-27 Serum-free Supplements (50X), liquid 10 ml 17504-044

B-27 Supplement Minus AO (50X), liquid 10 ml 10889-038

B-27 Supplement Minus Vitamin A (50X), liquid 10 ml 12587-010

G-5 Supplement (100X), liquidG-5 Supplement is for the growth and expression of glial cells (normal and tumor) of astrocytic phenotype .For product formulation please refer to the tables on page 162 .Recommended storage conditions: –5°C to –20°C .


1 ml 17503-012

N-2 Supplement (100X), liquidN-2 Supplement is for the growth and expression of post-mitotic neurons and tumor cells of neuronal phenotype .For product formulation please refer to the tables on page 162 .Recommended storage conditions: –5°C to –20°C .


5 ml 17502-048

Brain-Derived Neurotrophic Factor (BDNF), Human, RecombinantBrain-Derived Neurotrophic Factor (BDNF), Human, Recombinant is prepared from E. coli, and is supplied lyophilized from distilled water with no additives .Applications: Maintenance of motor neurons and the differentiation of both cholinergic and dopaminergic neurons . Performance and quality testing: Determined to be >96% pure by SDS-PAGE . Functional performance is verified by dose-dependent induction of choline acetyl transferase activity in primary septal cell cultures .Recommended storage conditions: –20°C . Notes: Avoid repeated freezing and thawing . Do not store in glass or in dilute solution . Membrane-filtered through 0 .22 µm filter .


5 µg 10908-010



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Nerve Growth Factor 2.5S (NGF 2.5S), Murine, NaturalNGF 2 .5S, Murine, Natural a ~26kDa subunit of the 7S NGF complex, is the 2 .5S subunit responsible for most of the biological activities of NGF . The 2 .5S subunit promotes nerve growth and maintenance of adrenergic neurons in vitro . This preparation of NGF 2 .5S is purified from the submaxillary glands of male mice and is supplied at a concentration of 100 µg/ml in 10 mM sodium acetate (pH 5 .0) .Applications: Induction of neurite outgrowth; maintenance of neural cell viability in culture; affects cellular morphology and differentiation; stimulates protein tyrosine .Performance and quality testing: Determined to be >95% pure by SDS-PAGE . Functionality is verified in a neurite outgrowth assay using PC12 cells .Recommended storage conditions: –20°C .Notes: Avoid repeated freezing and thawing . Do not store in dilute solution .


10 µg 13257-019

Nerve Growth Factor 7S (NGF 7S), Murine, NaturalNGF 7S, Murine, Natural, is a 130–140-kDa complex that regulates the development and maintenance of sympathetic and some sensory neurons . Murine natural NGF 7S is active on human, mouse, rat, and chicken cells . The NGF 7S is supplied lyophilized from PBS .Applications: Studies of nerve cell growth and differentiation, and biochemical studies of the basis of the various rapid and delayed biological effects of NGF . It can also be used as a medium supplement to support the growth or survival of neuronal cells in culture .Performance and quality testing: Determined to be >95% pure by SDS-PAGE . Functional performance is verified by measuring neurite outgrowth in chick embryo dorsal root ganglia .Recommended storage conditions: –20°C .Notes: Avoid repeated freezing and thawing . Do not store in dilute solution . Membrane-filtered through a 0 .22 µm filter .


100 µg 13290-010

ViraPower™ Lentiviral Expression SystemThe ViraPower™ Lentiviral Expression System readily transduces both dividing and non-dividing cell types (neurons), enabling analysis of long-term stable gene expression . Use this system to broaden your ability to analyze gene expression beyond the limitations of traditional transfection reagents or other retroviral systems .

Invitrogen is the only commercial supplier of an inducible and promoterless lentiviral expression system .


ViraPower™ Lentiviral Directional TOPO® Expression Kit 1 kit K4950-00

ViraPower™ Lentiviral Gateway® Expression Kit 1 kit K4960-00

ViraPower™ Zeo Lentiviral Gateway® Expression Kit 1 kit K4980-00

ViraPower™ UbC Lentiviral Gateway® Expression Kit 6 µg K4990-00

ViraPower™ T-Rex Lentiviral Expression System 1 kit K4965-00

ViraPower™ Promoterless Lentiviral Gateway® Expression System 1 kit K5910-00

For more information on the ViraPower™ Lentiviral Expression System, please visit our website at www .invitrogen .com .


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Stem Cell Research ProductsNeurobasal™-A Medium (1X), liquidNeurobasal™-A Medium is for long-term growth and viability of rat postnatal and adult hippocampal and cortical neurons of the CNS .2



500 ml 10888-022

References1 . Brewer, G .J ., Torricelli, J ., Evege, E .K ., and Price, P . (1994) FOCUS® 16, 6 .2 . Brewer, G .J . (1997) J. Neurosci. Methods 71, 145 .3 . Brewer, G ., Price, P . (2001) Protocols for Neural Cell Culture, 3rd Ed ., Ed .: S . Federoff

and A . Richardson, Humana Press, 255–264 .4 . Hermann, A . et al . (2004) J Cell Sci. 117, 4411–4422 .

AIM V® Media (research grade)Performance testing: Performance tested in a growth assay using Jurkat cells (ATCC Jurkat T1B-152) .Viral testing: Raw material components of human origin are nonreactive, at the donor level, for Hepatitis B surface antigen (HBsAG), antibody to Hepa titis C virus (anti-HCV), antibodies to Human Immunodeficiency Viruses 1 and 2 (anti-HIV 1 and 2), and syphilis, as determined by currently approved testing procedures . Raw material components of human origin are also tested for alanine aminotransferase (ALT) enzyme, and found to be within normal limits . Where possible, such components are further |processed by heat treatment in liquid form at 60°C for 10 hours by validated methods demonstrated to inactivate adventitious viral contam inants (performed by our raw material vendors) .

AIM V® Medium, liquid (research grade, with HSA)Recommended storage conditions: 2°C to 8°C, in the dark .


500 ml 12055-091

1,000 ml 12055-083

Contains L-glutamine, streptomycin sulfate at 50 µg/ml, and gentamicin sulfate at 10 µg/ml .

AIM V® Medium, liquid (research grade, with BSA)Recommended storage conditions: 2°C to 8°C, in the dark .


500 ml 31035-025

Contains AlbuMAX® supplement (bovine serum albumin) substituted for human serum albumin, L-glutamine, streptomycin sulfate at 50 µg/ml, and gentamicin sulfate at 10 µg/ml .

B-27 Supplement Minus AO (50X), liquidB-27 Supplement Minus AO is the complete B-27 Supplement without 5 antioxidants (vitamin E, vitamin E acetate, superoxide dismutase, catalase, and glutathione) that would interfere with studying free-radical damage to neurons .


10 ml 10889-038

B-27 Supplement Minus Vitamin A (50X), liquidB-27 Supplement Minus Vitamin A is the complete B-27 Supplement without Vitamin A . The deletion of vitamin A in this supplement prevents neural differentiation of stem cells .Recommended storage conditions: –5°C to –20°C .

References1 . Brewer, G .J ., Torricelli, J .R ., Evege, E .K ., and Price, P .J . (1993) J. Neurosci. Res. 35,

567 .2 . Brewer, G .J ., Torricelli, J .R ., Evege, E .K ., and Price, P .J . (1994) FOCUS® 16, 6 .3 . Brewer, G ., Price, P . (2001) Protocols for Neural Cell Culture, 3rd Ed ., Ed .: S . Federoff

and A . Richardson, Humana Press, 255–264 .4 . Hermann, A . et al . (2004) J Cell Sci. 117, 4411–4422 .


10 ml 12587-010

N-2 Supplement (100X), liquidN-2 Supplement is for the growth and expression of post-mitotic neurons and tumor cells of neuronal phenotype .For product formulation please refer to the tables on page 162 .Recommended storage conditions: –5°C to –20°C .


5 ml 17502-048

Neurobasal™ Medium (1X), liquidNeurobasal™ Medium is for long-term growth of fetal hippocampal neu-rons and many other neurons of the CNS .1


500 ml 21103-049



4



AIM V® Media (therapeutic grade)Performance testing: Performance tested in a growth assay using Jurkat cells (ATCC Jurkat T1B-152) .Viral testing: Raw material components of human origin are non reactive, at the donor level, for Hepatitis B surface antigen (HBsAG), antibody to Hepatitis C virus (anti-HCV), antibodies to Human Immunodeficiency Viruses 1 and 2 (anti-HIV 1 and 2), and syphilis, as determined by currently approved testing procedures . Raw material components of human origin are also tested for alanine aminotrans ferase (ALT) enzyme, and found to be within normal limits . Where possible, such components are further processed by heat treatment in liquid form at 60°C for 10 hours by validated methods demonstrated to inactivate adventitious viral contaminants (performed by our raw material vendors) .Intended uses: for human ex vivo tissue and cell culture processing applications .

AIM V® Medium, liquid (therapeutic grade)Recommended storage conditions: 2°C to 8°C, in the dark .Intended uses: For human ex vivo tissue and cell culture processing applications .Notes: An AIM V® Medium Master File has been submitted to the FDA . Per-mission to cross-reference the Master File may be obtained by contacting Invitrogen Technical Support or your local Sales Representative .


1,000 ml 087-0112DK

10 L 087-0112BK

Contains L-glutamine, 50 µg/ml streptomycin sulfate, and 10 µg/ml gentamicin sulfate .

MesenPRO RS™ MediumMesenPRO RS™ is a reduced-serum (2%) medium specif ical ly formulated to support the growth of human mesenchymal stem cells (MSCs) in culture . MesenPRO RS™ Medium consistently improves MSC expansion compared with conventional serum-supplemented media . Using MesenPRO RS™ Medium, MSCs can be expanded for multiple passages while maintaining their multipotential phenotype (i .e ., differentiation into osteogenic, chondrogenic, and adipogenic lineages) .

MesenPRO RS™ consists of basal liquid medium (MesenPRO RS™ Basal Medium), and frozen supplement (MesenPRO RS™ Growth Supplement), which is added at the time of use .Applications: Formulated for the growth and expansion of mesenchymal stem cells .Recommended storage conditions: MesenPRO RS™ Basal Medium, 2°C to 8°C, in the dark; MesenPRO RS™ Growth Supplement, –5°C to –20°C, in the dark . Both the Basal Medium and Growth Supplement are guaranteed stable for 12 months when properly stored .Intended use: In vitro diagnostic (IVD) .Notes: MesenPRO RS™ Medium has been developed for culturing MSCs at greater than clonal densities . MesenPRO RS™ complete medium (basal, supplement, and glutamine) is stable for 15 days when stored at 2°C to 8°C, in the dark .


MesenPRO RS™ Medium* 1 kit 12746-012

Related products

Gentamicin Reagent Solution (10 mg/ml), liquid 100 ml 15710-049

L-Glutamine-200 mM (100X), liquid 100 ml 25030-024

Dulbecco’s Modified Eagle Medium (DMEM) low glucose (1X), liquid 500 ml 11880-028

Dulbecco’s Phosphate Buffered Saline (DPBS) without calcium, magnesium, or phenol red (1X), liquid 500 ml 14190-094

TrypLE™ Express (1X), liquid, without phenol red 500 ml 12604-021

STRO-1 (host: mouse; clone: Stro-1) (Zymed®) 100 μl 39-8401

* This product is shipped in two parts, each part having separate storage requirements and separate catalogue numbers: MesenPRO RS™ Basal Medium (500 ml, Cat . no . 12747-010) and MesenPRO RS™ Growth Supplement (10 ml, Cat . no . 12748-018) . The component catalogue numbers are not available for purchase separately .


118


StemPro® hESC SFM StemPro® hESC SFM, developed for human embryonic stem cell culture, is a fully-defined, serum- and feeder-free medium (SFM) specially formulated for the growth and expansion of human embryonic stem cells (hESCs) . StemPro® hESC SFM has been extensively tested and proven to maintain pluripotency in a growing list of hESC lines including BG01, BG02, BG03, H1, H9, and BG01V . In addition, StemPro® hESC SFM has been used to scale up production of hESCs to over 1 × 1010 cells . StemPro® hESC SFM has been shown to support hESC growth for >50 passages without any signs of karyotypic abnormalities and to maintain the ability of hESCs to differentiate into all three germ line lineages .

StemPro® hESC SFM consists of StemPro® hESC Supplement, DMEM/F-12 with GlutaMAX™, and Bovine Serum Albumin 25% (BSA) .

This product is shipped in two parts with separate storage requirements: 1 . StemPro® hESC Supplement (10 ml, Cat . no . A1000601); 2 . Bovine Serum Albumin 25% (BSA) (40 ml, Cat . no . A1000801) and

DMEM/F-12 with GlutaMAX™ (500 ml, Cat . no . 10565-018) . These components, with the exception of DMEM/F12 with GlutaMAX™, are not available for purchase separately .Applications: Formulated for the growth and expansion of human embryonic stem cells .Recommended storage conditions: StemPro® hESC Supplement, –5°C to –20°C, in the dark; DMEM/F-12 with GlutaMAX™ and Bovine Serum Albumin 25% (BSA), 2°C to 8°C (protect from light) . These components are guaranteed stable for 12 months when properly stored .Intended use: For research use only . Notes: Additional components required for complete medium: FGF BASIC (full length) REC HU (Cat . no . PHG0261), 2-Mercaptoethanol (Cat . no . 31350-010) .StemPro® hESC SFM Complete Medium (Supplement, DMEM/F-12, and BSA) is stable for 1 day when stored in the dark from 2°C to 8°C . We recommend reconstituting minimal volumes of working complete medium (for the day), just prior to use and pre-equilibrating to temperature and gases .


StemPro® hESC SFM 1 kit A1000701

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2-Mercaptoethanol 20 ml 31350-010

BG01V/hOG Cells 1 vial R7799-105

Collagenase Type IV 1 g 17104-019

DPBS, without calcium, magnesium, or phenol red 500 ml 14190-094

FGF BASIC (full length) REC HU 100 μg PHG0261

Geltrex™ Reduced Growth Factor Basement Membrane Matrix 5 ml 12760-021

SSEA1 (host: mouse; clone: MC-480) 100 µg 41-1200

SSEA3 (host: rat; clone: MC631) 100 µg 41-4400

SSEA4 (host: mouse; clone: MC813-70) 100 µg 41-4000

StemPro® EZPassage™ 1 box, 10 units 23181-010

TRA-1-60 (host: mouse; clone: cl .A) 100 µg 41-1000

TRA-1-81 (host: mouse; clone: cl .26) 100 µg 41-1100

TrypLE™ Express Stable Trypsin Replacement Enzyme without phenol red 100 ml 12604-013



4



StemPro®-34 SFM (1X), liquidStemPro®-34 SFM is a serum-free medium specifically formulated to support the development of human hematopoietic cells in culture .1

StemPro®-34 SFM can be used to study hematopoietic cells from bone marrow, peripheral blood, or neonatal cord blood . StemPro®-34 SFM is performance-tested on normal human bone marrow . It is manufactured with components of human, recombinant, or synthetic origin .

StemPro®-34 SFM consists of a basal liquid medium and frozen supplement (StemPro®-Nutrient Supplement) which is added at the time of use .Recommended storage conditions: medium, 2°C to 8°C; supplement, –5°C to –20°C .Notes: An intended use disclosure is required for purchase of StemPro®-34 SFM . For further regulatory information on the uses of this product, please contact theTech-LineSM .

References1 . Daley, J .P ., Dadey, B .M ., Wysocki, M .G ., Caligiuri, M .A ., and Biddle, W .C . (1996)

FOCUS® 18, 62 . 2 . Taylor, M . et al . (2001) Blood 98, 1195–1199 .3 . Szilvassy, S . et al . (1999) Blood 93, 1557–1566 .4 . Delehanty, L . et al . (2003) Blood 101, 1744–1751 .5 . Kirshenbaum, A . et al . (1999) Blood 94, 2333–2342 .6 . Szilvassy, S ., Meyerrose, T ., and Grimes, B . (2000) Blood 95, 2829–2837 .7 . Yahata, T . et al . (2003) Blood 101, 2905–2913 .8 . Wang, J . et al . (2003) Blood 101, 2924–2931 .9 . Kolbus, A . et al . (2003) Blood 102, 3136–3146 .10 . Goyama, S . et al . (2004) Blood 104, 3558–3564 .


500 ml 10639-011

DMEM/F-12 (1X), liquid, 1:1A GlutaMAX™-I medium . Recommended storage conditions: 2°C to 8°C .Intended uses: In vitro diagnostic (IVD) .For product formulation see page 91 .


500 ml 31331-028

10 × 500 ml (case) 31331-093

Contains GlutaMAX™-I substituted on a molar equivalent basis for L-glutamine, but no HEPES buffer .

KnocKout™ Serum ReplacementKnocKout™ Serum Replacement (KnocKout™ SR) is a defined, serum-free formulation optimized to grow and maintain undifferentiated ES cells in culture .1 It directly replaces FBS in existing protocols . The performance of KnocKout™ SR is enhanced when used with KnocKout™ DMEM . Product performance is confirmed by evaluation of its ability to support the growth of undifferentiated D3 ES cell colonies on inactivated mouse embryonic fibroblasts .Recommended storage conditions: –5°C to –20°C, in the dark .

Reference1 . Goldsborough, M ., Tilkins, M . L ., Price, P ., Lobo-Alfonso, J ., Morrison, J ., Stevens,

M ., Meneses, J ., Pederson, R ., Koller, B ., and Latour, A . (1998) FOCUS® 20, 8 .

Related Reading1 . Amit, M ., Carpenter, M .K ., Inokuma, M ., Chiu, C ., Harris, C .P ., Waknitz, M .A .,

Itskovitz-Eldor, J . and Thomson, J .A . Clonally Derived Human Embryonic Stem Cell Lines Maintain Pluripotency and Proliferative Potential for Prolonged Periods of Culture . Developmental Biology 227, 271-278, (2000) .

2 . Kawasaki, H ., Suemori, H ., Mizuseki, K ., Watanabe, K ., Urano, F ., Ichinose, H ., Haruta, M ., Takahashi, M ., Yoshikawa, K ., Nishikawa, S ., Nakatsuji, N . and Sasai, Y . Genera-tion of dopaminergic neurons and pigmented epithelia from primate ES cells by stromal cell-derived inducing activity . Proc. Natl. Acad. Sci. 99;3, 1580–1585, (2002) .

3 . Lebkowski, J .S ., Gold, J ., Xu, C ., Funk, W ., Chiu, C ., and Carpenter, M .K . Human Embryonic Stem Cells: Culture, Differentiation, and Genetic Modification for Regenerative Medicine Applications . Cancer Journal 7(2), S83-S93, (Nov-Dec 2001) .

4 . Nakayama, N ., Lee, J . and Chiu, L . Vascular endothelial growth factor synergistically enhances bone morphogenetic protein-4-dependent lymphohematopoietic cell generation from embryonic stem cells in vitro. Blood 1;95(7), 2275-2283 (2000) .

5 . Schuldiner, M ., Yanuka, O ., Itskovitz-Eldor, J ., Melton, D .A . and Benvenisty, N . Effects of eight growth factors on the differentiation of cells derived from human embryonic stem cells . Proc. Natl. Acad. Sci. USA 97;21, 11307-11312 (2000) .

6 . Amit, M ., Shariki, C ., Margulets, V . and Itskovitz-Eldor, J . (2004) Biol Reprod. 70(3), 837–845 .

7 . Cheng, J ., Dutra, A ., Takesono, A ., Garrett-Beal, L . and Schwartzberg, P .L . (2004) Genesis 39, 100-104 .

8 . Rosler, E .S ., Fisk, G .J ., Ares, X ., Irving, J ., Miura, T ., Rao, M .S . and Carpenter, M .K . (2004) Dev Dyn. 229(2), 259–74 .

9 . Schulz, T ., et al . (2004) Stem Cells 22, 1218–1238 .10 . Wang, L ., et al . (2005) Blood Jun 15;105(12):4598-603 . Epub 2005 Feb 17 .


500 ml 10828-028

Related products PageGeneticin® Selective Antibiotic . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .153TrypLE™ Cell Dissociation Products . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .159


120


KnocKout™ DMEMKnocKout™ DMEM is a basal medium optimized for growth of un-differ-entiated ES cells .1 The osmolarity is optimized to approximate that of mouse embryonic tissue . Recommended storage conditions: 2°C to 8°C, in the dark .Notes: Prior to use, KnocKout™ DMEM must be supplemented with either KnocKout™ Serum Replacement or FBS . Formulated without L-glutamine for increased stability ; add 5 ml of 200 mM glutamine or 5 ml of GlutaMAX™-I Supplement per 500 ml of medium prior to use .

Reference1 . Goldsborough, M ., Tilkins, M . L ., Price, P ., Lobo-Alfonso, J ., Morrison, J ., Stevens,

M ., Meneses, J ., Pederson, R ., Koller, B ., and Latour, A . (1998) FOCUS® 20, 8 .


500 ml 10829-018


Related products PageLipofectamine™ 2000 Transfection Reagent . . . . . . . . . . . . . . . . . . . . . . . . .132TrypLE™ Cell Dissociation Products . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .159



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Geneticin® Selective AntibioticA selection agent commonly used for the selection of mammalian, plant, or yeast cells . This selection reagent is available in powder form or as a convenient aseptic liquid, provided at 50 mg/ml active concentration for consistent potency . Geneticin® agent is an analog of neomycin sulfate that interferes with the function of 80S ribosomes and protein synthesis in eukaryotic cells . The Tn5 or Tn601 aminoglycoside phosphotransferase gene (neo) confers resistance . When evaluating G-418 products, it is important to consider more than just potency . It is critical to look at purity and work-ing range . Taken together, these three characteristics provide a true evaluation of the effectiveness of the G-418 preparation (sidebar) .

To demonstrate the higher standards of the Geneticin® agent, we recently compared three aspects of Geneticin® reagent to various lots of G-418 from two different suppliers (see table below) . In all of the parameters, Geneticin® reagent proved to be more consistent .

Geneticin® is available in both solid (powder) and liquid formats . The higher-potency liquid eliminates the time and effort spent producing working stocks from powder . Each lot is supplied at 50 mg/ml active concentration for consistent potency . Each lot is tested in an in vitro cell culture assay to ensure performance and validate label potency .Formulation: C20H40N4O10•2H2SO4

Formula weight: 496.6•196.1 g/molContents and storage: Liquid Geneticin®: Supplied as a 50 mg/ml solution; store at –20°C, or 2°C–8°C; guaranteed for 6 months when stored correctly . Powder Geneticin®: store at room temperature prior to reconstitution, and at 2°C to 8°C after reconstitution; guaranteed stable for 6 months when stored correctly .


Geneticin® Selective Antibiotic, powder 1 g 11811-023 5 g 11811-031 10 g 11811-064 25 g 11811-098

Geneticin® Selective Antibiotic, liquid 20 ml (50 mg/ml) 10131-019 100 ml (50 mg/ml) 10131-027

Comparison of G-418 Specifications of Geneticin® vs. Products from Suppliers A and BSpecification Invitrogen™ Supplier A Supplier B The Geneticin® advantage

Purity (HPLC) >90–93% 66–75% 65–82% Significantly higher purity

Potency claimed (µg/mg) 718–735 712–724 673–735 Most stringent potency assay

Potency upon retest (µg/mg) 718–735 640–659 621–677 Consistently higher actual potency

ED50 (µg/ml) 2,450–2,700 1,350–3,100 600–2,350 Reliable selection, lot to lot

Comparison of Geneticin® specifications with alternative G-418 suppliers . Multiple lots of G-418 from two different sup-pliers were analyzed according to Invitrogen raw material QC protocols for Geneticin® . Results obtained with G-418 from Supplier A and Supplier B displayed wide variation on all measured parameters . Discrepancies between retested potency values and those claimed by other suppliers may be due to differences in G-418 reference lots and assay procedures .

Look for consistency in these three G-418 specifications:

Purity: Purity of Geneticin® selection agent is consistently greater than 90%, as deter-mined by HPLC—far higher than any other supplier . This means, you can generally use 15–30% lower concentrations of Geneti-cin® agent than other G-418 products to achieve comparable selection results .

Potency: This is one of the most misunderstood and misused criteria for comparing and assessing product qual-ity . Potency is a prokaryotic selection parameter . Higher-potency G-418 is most effective for mammalian cell selection when it is also high in purity . Gentamicins that commonly contaminate G-418 prep-arations will add to potency in bacterial assays . However, they have no effect on mammalian cell selectivity . Furthermore, other bacterial contaminants in impure G-418 may be toxic to mammalian cells in culture, consequently lowering the ED50 value .

ED50 Assay: This is a true measure of eukary-otic growth selectivity as determined on reference NIH3T3 cells . Higher purity generally translates to higher ED50 values . Toxic impurities lower the ED50 value, which means a more narrow working range of antibiotic selection . A consistent ED50 from a supplier assures performance reproducibility from lot to lot in mamma-lian cell culture applications .


122


Blasticidin S HCl, powderBlasticidin is a nucleoside antibiotic from Streptomyces griseochromogenes . It is a potent translational inhibitor in both prokaryotic and eukaryotic cells . Resistance to blasticidin is conferred by the bsd gene product from Aspergillus terreus . Blasticidin has a fast, potent mode of action, causing rapid cell death at low antibiotic concentrations . E. coli strains are generally sensitive to concentrations of 50 µg/ml, while mammalian cells are sensitive to concentrations as low as 2 to 10 µg/ml . Cell death occurs rapidly, and blasticidin-resistant stable mammalian cell lines can be generated in less than one week, at low concentrations .Recommended storage conditions: –20°C .


50 mg R210-01

Hygromycin BHygromycin B is an aminoglycosidic antibiotic that inhibits protein synthesis by disrupting translocation and promoting mistranslation at the 80S ribosome . Because it uses a different mode of action than Geneticin®, blasticidin S, or Zeocin™, it’s perfect for dual-selection experiments when used in conjunction with another selection agent . Provided in solution at a concentration of 50 mg/ml .Recommended storage conditions: 4°C .


20 ml 10687-010

GlutaMAX™-I SupplementExtremely stable in aqueous solution . See Appendix I, page 202 .

200 mM supplied in 0 .85% NaCl .Recommended storage conditions: –5°C to –20°C .

Reference1 . Christie, A . and Butler, M . (1994) FOCUS® 16, 9 .


100 ml 35050-038

20 × 100 ml (case) 35050-087

Contains the dipeptide L-alanyl-L-glutamine .

TrypLE™ Express, Stable Trypsin Replacement Enzyme (1X), liquidA pure enzyme for gentler cell dissociation—ideal for stem cells . This

recombinant enzyme is an alternative to animal trypsin for the dissociation of

adherent mammalian cells . See page 151 for ordering information .

Fetal Bovine Serum, ES cell-qualified

Origin: United StatesSpecially tested for the ability to sustain undifferentiated cellular morphology of embryonic stem cells .Endotoxin level: ≤50 EU/ml .Hemoglobin level: ≤25 mg/dl .Recommended storage conditions: –5°C to –20°C .Intended use: In vitro diagnostic (IVD) .


100 ml 16141-061

500 ml 16141-079

Origin: Countries meeting USDA importation requirementsSpecially tested for the ability to sustain undifferentiated cellular morphol-ogy of embryonic stem cells .Endotoxin level: ≤50 EU/ml .Hemoglobin level: ≤25 mg/dl .Recommended storage conditions: –5°C to –20°C .Intended use: In vitro diagnostic (IVD) .


100 ml 10439-016

500 ml 10439-024

CO2-Independent Medium (1X), liquidA non-HEPES proprietary medium suitable for supporting cell growth for a variety of epithelial, fibroblast, and lymphoid cell lines in atmospheric conditions .1 It is ideal for transporting cells or tissue and for the handling of mouse embryos in atmospheric conditions . Not intended for CO2-based mouse embryo culture .Recommended storage conditions: 2°C to 8°C, in the dark .

Reference1 . Battista, P .J . and Weiss, S .A . (1991) FOCUS® 13, 110 .


500 ml 18045-054

10 × 500 ml 18045-070





4



Basic Fibroblast Growth Factor (bFGF), Human, RecombinantbFGF, Human, Recombinant, also known as Heparin Binding Growth Factor (HBGF)-2, is a 17kDa protein supplied lyophilized with no additives . Basic FGF is structurally similar to acidic FGF, but is functionally different .Applications: Studies of angiogenesis; mitogenesis of fibroblasts; neurite outgrowth in PC-12 cells; receptor binding; tyrosine protein kinase phosphorylation .Performance and quality testing: Determined to be >95% pure by SDS-PAGE and N-terminal sequencing . Functional performance is verified by thymidine incorporation into serum-starved Balb/c 3T3 cells .Recommended storage conditions: –20°C .Notes: Avoid repeated freezing and thawing . Do not store in dilute solution . Membrane-filtered through a 0 .22 µm filter .


100 µg EGF Recombinant Human PHG0311 10 µg EGF Recombinant Human PHG0314 25 µg EGF Recombinant Human PHG0315 100 µg EGF Recombinant Human (Liquid Form) PHG0311L

Insulin-like Growth Factor-I (IGF-I), Human, RecombinantIGF-I, Human, Recombinant, is prepared from E. coli and supplied lyoph ilized from water .Applications: Receptor binding, transmembrane signaling, protein phosphorylation, and epidermal cell proliferation studies .Performance and quality testing: Determined to be >97% pure by SDS-PAGE . Functional performance is verified by thymidine incorporation into Balb/c 3T3 cells .Recommended storage conditions: –20°C .Notes: Avoid repeated freezing and thawing . Do not store in glass or in dilute solution . Membrane-filtered through a 0 .22 µm filter .


10 µg PHG9074

Platelet-derived Growth Factor BB (PDGF BB), Human, RecombinantPDGF BB, Human, Recombinant, is supplied lyophilized from water .Applications: Receptor binding studies; studies of transmembrane signaling and protein phosphorylation; and fibroblast proliferation studies .Performance and quality testing: PDGF BB is determined to be >97% pure by SDS-PAGE . Functional performance is verified by thymidine incorporation into confluent Balb/c 3T3 cells .Recommended storage conditions: –20°C .Notes: Avoid repeated freezing and thawing . Do not store in glass or in dilute solution . Membrane-filtered through a 0 .22 µm filter .


100 µg PDGF-BB Recombinant Mouse PMG0041 1mg PDGF-BB Recombinant Mouse PMG0043 10 µg PDGF-BB Recombinant Mouse PMG0044 25 µg PDGF-BB Recombinant Mouse PMG0045

Vascular-Endothelial Products

Human Endothelial-SFMHuman Endothelial-SFM is a basal serum-free growth medium developed to support the isolation and expansion of human umbilical venous and arterial endothelial cells .1–4

Medium requires supplementation with bFGF (20 ng/ml), EGF (10 ng/ml), and human plasma fibronectin (10 µg/ml) .Recommended storage conditions: 2°C to 8°C, in the dark .Notes: A Human Endothelial-SFM Master File has been submitted to the FDA . Permission to cross-reference the Master File may be obtained by contacting Invitrogen Technical Support or your local Sales Representative .


124


References1 . Battista, P .J ., Bowen, H .J ., and Gorfien, S .F . (1995) FOCUS® 17, 10 .2 . Soderland, C ., Veres, J .S ., and Battista, P .J . (1995) FOCUS® 17, 14 .3 . Battista, P .J . and Soderland, C . (1995) FOCUS® 17, 106 .4 . Battista, P .J ., Soderland, C ., Tilkins, M .L ., and Gorgien, S .F . (1996) American

Biotechnology Laboratory 14, 34 .5 . Battista, P .J . and Soderland, C . (1998) Cell Biology: A Laboratory Handbook 1, 137 .


500 ml 11111-044


Basic Fibroblast Growth Factor (bFGF), Human, RecombinantbFGF, Human, Recombinant, also known as Heparin Binding Growth Factor (HBGF)-2, is a 17kDa protein supplied lyophilized with no additives . Basic FGF is structurally similar to acidic FGF, but is functionally different .Applications: Studies of angiogenesis; mitogenesis of fibroblasts; neurite outgrowth in PC-12 cells; receptor binding; tyrosine protein kinase phosphorylation .Performance and quality testing: Determined to be >95% pure by SDS-PAGE and N-terminal sequencing . Functional performance is verified by thymidine incorporation into serum-starved Balb/c 3T3 cells .Recommended storage conditions: –20°C .Notes: Avoid repeated freezing and thawing . Do not store in dilute solution . Membrane-filtered through a 0 .22 µm filter .


10 µg 13256-029

Fibronectin, Human PlasmaFibronectin is a large cell-surface and plasma protein that exhibits structural and adhesive properties in cell-associated fibrillar matrices . Fibronectin is one of the primary cell adhesion molecules . Human fibronectin is filtered (0 .2 µm) and supplied lyophilized .Performance and quality testing: Purity is determined by SDS-PAGE . Human fibronectin is assayed for its ability to support cell attachment and spreading, and screened to confirm absence of myco plasmas or bacterial contamination .Recommended storage conditions: –5°C to –20°C .Notes: Do not store in dilute solution . Avoid repeated freezing and thawing .


5 mg 33016-015



4



Cytogenetics ProductsQuality ControlAll MarrowMAX™, AmnioMAX™, and KaryoMAX® products have been stringently qualified by an independent, clinically certified cytogenetic laboratory using standardized cytogenetic protocols . This qualified line of products helps you attain reproducible, quality results in standard clinical cytogenetic protocols involving the analysis of either amniotic fluid cells, chorionic villus samples, peripheral blood lymphocytes, or bone marrow cells .Warning: Although the media and reagents are safe to use, the human peripheral blood used in procedures with these products may be ` infectious or hazardous to the user . Be sure to use proper handling, decontamination, and waste disposal techniques .

MarrowMAX™, AmnioMAX™, and KaryoMAX® Products—Certified for Consistency in CytogeneticsMarrowMAX™, AmnioMAX™, and KaryoMAX® products are specifically formulated by us and application tested by an independent laboratory to deliver clear, reproducible results in standard clinical cytogenetic protocols involving the analysis of amniotic fluid cells, chorionic villus samples, peripheral blood lymphocytes, or bone marrow cells .

The combination of the gibco® production process and strict quality control provides you with confidence that every lot of MarrowMAX™, AmnioMAX™, or KaryoMAX® products will support healthy cell morphology and rapid culture growth . Our goal is to help you obtain results that are easy to analyze and interpret .

MarrowMAX™ Bone Marrow MediumMarrowMAX™ Bone Marrow Medium is a fully supplemented medium developed specifically to support bone marrow and peripheral blood cell culture for in vitro cytogenetic analysis of hematological disease .Recommended storage conditions: –5°C to –20°C, in the dark .Intended use: In vitro diagnostic (IVD) .

This product is the subject of claims in US Patent No . 6,103,522 and equivalent foreign patents owned by the Fred Hutchinson Cancer Research Center . Purchase of this product conveys to the buyer only the non-transferable right under these patents to use the purchased amount of the product in research conducted by the buyer . The buyer cannot sell or otherwise transfer this product or any derivatives thereof to any third party . The buyer may not use this product or any derivatives thereof for any in vivo diagnostic or clinical use in humans . Fred Hutchinson Cancer Research Center and Invitrogen Corporation reserve all other rights, and this product may not be used in any manner other than as provided herein . In particular, without prior written agreement between the buyer and Invitrogen, no rights under the above patents are conveyed to the buyer to use the product in manufacturing, or to resell the product or components of the product as a stand-alone reagent or in a kit, whether or not such stand-alone reagent or such kits are resold for use in research . If the purchaser is not willing to accept the limitations of this limited use label license, Invitrogen is willing to accept return of the product with full refund . For information on purchasing a license to this product for purposes other than research, contact the Fred Hutchinson Cancer Research Center, 1100 Fairview Avenue N . C2M-027, Seattle, WA 98104 if such use is in vivo diagnostic or clinical use in humans, or Invitrogen Corporation, 1600 Faraday Avenue, Carlsbad, CA 92008 . Phone 760 .603 .7200 . Fax 760 .602 .6500 if such use is for the manufacture of a product for use in an ex vivo diagnostic or as a pharmaceutical product .


100 ml 12260-014

Quality Control Testing

Product Base cat. no.Bone

marrowPeripheral

bloodPrimary

amniocytesNumber of

coloniesMitotic colony Mitotic index

Banding resolution

MarrowMAX™ Bone Marrow Medium 12260 ● ● ●

AmnioMAX™-II Complete Medium 11269 ● ● ●

AmnioMAX™-C100 Complete Medium (system) 12558 ● ● ●

AmnioMAX™-C100 Basal Medium 17001 ● ● ●

AmnioMAX™-C100 Supplement 12556 ● ● ●

PB-MAX™ Karotyping Medium 12557 ● ● ●

KaryoMAX® Colcemid™ Solution in PBS 15212 ● ● ●

KaryoMAX® Colcemid™ Solution in HBSS 15210 ● ● ●

KaryoMAX® Giemsa Stain Stock Solution 10092 ● ●

Phytohemagglutinin 10576 ● ● ●

Colcemid™ is a trademark of CIBA Limited .


126


AmnioMAX™ Complete MediaAmnioMAX™ media are specifically formulated for the in vitro culture of human primary amniotic fluid cells . They are designed to optimize cell attachment and growth rates, and to provide high metaphase yields . Two different formulations are available, offering cytogenetics laboratories the ability to use a secondary cell culture medium to meet established laboratory protocols . AmnioMAX™-C100 Complete Medium consists of two components (basal medium and supplement), while AmnioMAX™-II Complete Medium is a s ingle -bott le formulat ion for added convenience .

AmnioMAX™-II Complete MediumAmnioMAX™-II Complete Medium is a second-generation formulation further optimized for cell attachment and increased pH stability .1 Supplied in a single bottle, it is complete with antibiotics and growth-promoting agents, and requires no further supplementation .Recommended storage conditions: –5°C to –20°C, in the dark .Intended use: In vitro diagnostic (IVD) .

Reference1 . Kuligowski, S ., Csirke, B ., Biddle, W .C ., Battista, P ., and Kielma, M . (1999) FOCUS®

21, 35 .


100 ml 11269-016

AmnioMAX™-C100 ProductsThe system consists of AmnioMAX™-C100 Basal Medium and AmnioMAX™-C100 Supplement . It is complete with antibiotics and growth-promoting agents, and requires no further supplementation .1 The medium and supplement are also available separately .Recommended storage conditions: medium, 2°C to 8°C, in the dark; supplement, –5°C to –20°C . Intended use: In vitro diagnostic (IVD) .

Reference1 . Biddle, W .C ., Kuligowski, S ., Lockwood, D .H ., Filby, J ., and Custer-Hagen, T .

(1992) FOCUS® 14, 80 .


AmnioMAX™-C100 Complete Medium, system 1 set 12558-011

AmnioMAX™-C100 Basal Medium, liquid 90 ml 17001-082 450 ml 17001-074

AmnioMAX™-C100 Supplement, liquid 15 ml 12556-015 75 ml 12556-023

KaryoMAX® ProductsWe offer a full line of karyotyping products designed to minimize setup time and to maximize lab efficiency .

KaryoMAX® medium is fully supplemented and specifically formulated to optimize cell growth, reducing culture time and days to harvest, and the mitotic index and banding resolution .

PB-MAX™ Karyotyping MediumPB-MAX™ Karyotyping Medium is a fully supplemented, ready-to-use RPMI-based medium designed for cultivation of lymphocytes from peripheral blood . It is performance tested on cells from whole blood . Every lot of PB-MAX™ Medium comes with a Certificate of Analysis detailing the mean mitotic index and mean banding resolution of chromosome 10 . Recommended storage conditions: –5°C to –20°C .Intended use: In vitro diagnostic (IVD) .


100 ml 12557-013

500 ml 12557-021

Contains fetal bovine serum, L-glutamine, and phytohemagglutinin .

KaryoMAX® Colcemid™ Solution, liquid (10 µg/ml), in HBSSKaryoMAX® Colcemid™ Solution is functionally tested as a blocking agent that arrests cells in metaphase by disrupting the cytoskeleton . It is performance tested on cells from whole blood . Every lot comes with a Certificate of Analysis detailing the mean mitotic index . Prepared in Hanks’ Balanced Salt Solution .Recommended storage conditions: 2°C to 8°C .Intended use: In vitro diagnostic (IVD) .



10 ml 15210-040



4



KaryoMAX® Colcemid™ Solution, liquid (10 µg/ml), in PBSKaryoMAX® Colcemid™ Solution is functionally tested as a blocking agent that arrests cells in metaphase by disrupting the cytoskeleton . It is performance tested on cells from whole blood . Every lot comes with a Certificate of Analysis detailing the mean mitotic index . Prepared in phosphate-buffered saline, but with no phenol red .Recommended storage conditions: 2°C to 8°C .Intended use: In vitro diagnostic (IVD) .



10 ml 15212-040

KaryoMAX® Giemsa Stain Stock SolutionFunctionally tested for consistent G-banding of chromosomes .Intended use: In vitro diagnostic (IVD) .


100 ml 10092-013

Related Products for Cytogenetics

Gurr Buffer Tablets (pH 6.8)Gurr Buffer Tablets offer a convenient way to prepare a liter of buffer at pH 6 .8 for dilution of Giemsa stain . Simply add a liter of distilled water to a Gurr Buffer Tablet and stir until the tablet is dissolved .


50 × 1 L 10582-013

Phytohemagglutinin (M Form) (PHA), liquidPhytohemagglutinin is a liquid extract of the red kidney bean Phaseolus vulgaris . This extract is capable of inducing blastogenesis in vitro in various mammalian mononuclear cells . Concentration: Because of the undefined nature of this extract, we are not able to assign weight/volume values on the active material in our final product .Performance and quality testing: Each lot is assayed in a performance test system .Recommended storage conditions after rehydration: –5°C to –20°C .Attention: This material is hazardous . All persons using this product should review the Material Safety Data Sheet before handling .


10 ml 10576-015

Potassium Chloride Solution, 0.075 MA premixed 0 .075 M hypotonic solution .

Enlarges cel ls and faci l i tates the spreading of metaphase chromosomes for subsequent analysis .Recommended storage conditions: 2°C to 8°C .Intended use: In vitro diagnostic (IVD) .


4 × 100 ml (case) 10575-090


128


Antibiotic-Antimycotic (100X), liquidSpectrum: Bacteria, fungi, and yeasts .Recommended storage conditions: –5°C to –20°C .Attention: This material is hazardous . All persons using this product should review the Material Safety Data Sheet before handling .


20 ml 15240-096

100 ml 15240-062

20 × 100 ml (case) 15240-112

Contains 10,000 units of penicillin (base), 10,000 µg of streptomycin (base), and 25 µg of amphotericin B (Fungizone™ Antimycotic)/ml in 0 .85% saline .

Fungizone™ Antimycotic, liquidSpectrum: Fungi and yeasts . Recommended concentration: 0 .25 to 2 .5 µg/ml . Recommended storage conditions: –5°C to –20°C . Attention: This material is hazardous . All persons using this product should review the Material Safety Data Sheet before handling .


20 ml 15290-018 50 ml 15290-026

Contains 250 µg of amphotericin B and 205 µg of sodium deoxycholate per ml as a solubilizer in distilled water .

Gentamicin Reagent Solution (10 mg/ml), liquidSpectrum: Gram-positive and gram-negative bacteria .Recommended concentration: 0 .5 to 50 µg/ml .Recommended storage conditions: 15°C to 30°C .Attention: This material is hazardous . All persons using this product should review the Material Safety Data Sheet before handling .


10 ml 15710-064

10 × 10 ml (case) 15710-072

Contains 10 mg/ml gentamicin sulfate in distilled water .

Quantity Cat. no.

10 ml 15750-060

10 × 10 ml (case) 15750-078

Contains 50mg/ml gentamicin sulfate in distilled water .

Fungizone™ is a trademark of E .R . Squibb & Sons, LLC .

Penicillin-Streptomycin, liquidSpectrum: Gram-positive and gram-negative bacteria .Recommended storage conditions: –5°C to –20°C .Attention: This material is hazardous . All persons using this product should review the Material Safety Data Sheet before handling .


20 ml 15140-148

100 ml 15140-122

20 × 100 ml (case) 15140-163

Contains 10,000 units of penicillin (base) and 10,000 µg of streptomycin (base) per ml, utilizing penicillin G (sodium salt) and streptomycin sulfate in 0 .85% saline .

Quantity Cat. no.

100 ml 15070-063

Contains 5,000 units of penicillin (base) and 5,000 µg of streptomycin (base) per ml, utilizing penicillin G (sodium salt) and streptomycin sulfate in 0 .85% saline .



4



Collagenase, lyophilized (Clostridiopeptidase A)Prepared from Clostridium histolyticum .E.C. Number: 3 .4 .2 .4 .3 .Activity: 125 to 200 units/mg weight . One unit will liberate 1 µmol amino acid from collagen, expressed as L-leucine, in 18 hr at 37°C .Recommended storage conditions: 2°C to 8°C .


500 mg 17018-029

GlutaMAX™-I Supplement200 mM, supplied in 0 .85% NaCl .

Extremely stable in aqueous solution . See Appendix I, page 202 .Recommended storage conditions: –5°C to –20°C .

Reference1 . Christie, A . and Butler, M . (1994) FOCUS® 16, 9 .


100 ml 35050-061

20 × 100 ml (case) 35050-079

Contains the dipeptide L-alanyl-L-glutamine .

L-Glutamine-200 mM (100X), liquidSupplied at 29 .2 mg/ml in 0 .85% NaCl .pH: 4 .7 to 6 .0 .Recommended storage conditions: –5°C to –20°C .Intended use: In vitro diagnostic (IVD) .


20 ml 25030-149

100 ml 25030-081

20 × 100 ml (case) 25030-123

RPMI Medium 1640 (1X), liquid (Folate-free)Recommended storage conditions: 2°C to 8°C .


500 ml 27016-021

Contains L-glutamine, but no folic acid .

Sodium Bicarbonate Solution, 7.5% (w/v)Recommended storage conditions: 15°C to 30°C .


100 ml 25080-060

20 x 100 ml 25080-102

Water, distilledMembrane filtered and endotoxin screened .Recommended storage conditions: 15°C to 30°C .


100 ml 15230-071

20 × 100 ml (case) 15230-196

500 ml 15230-162

10 × 500 ml (case) 15230-788

1,000 ml 15230-147


130


Overview of Transfection, Selection, and Protein Delivery MethodsThe easy, fast, and reliable introduction of DNA, siRNA, RNA, and protein into mammalian cells is crucial to experimental results . Improvements in transfection reagent technology have allowed researchers to quickly discover novel genes and rapidly elucidate gene function . Transfection: Reagent-mediated transfection is a fast, simple, and reproducible means for easily introducing DNA, RNA, siRNA, or oligonucleotides into eukaryotic cells (Figure 1) . Transfections using reagents such as Lipofectamine™ LTX, FreeStyle™ MAX, and Lipofectamine™ 2000 are superior to older methods of transfection like DEAE-dextran and calcium phosphate precipitation for both transient protein expression and the generation of high-expression stable cell lines . Life Technologies™ transfection reagents offer consistent and reliable high performance . These reagents allow highly efficient transfection of a broad range of cell types, including adherent, suspension, insect, and primary cultures (Table 1) . All of Invitrogen’s transfection reagents include streamlined protocols for maximum convenience and ease of use . Many reagents include protocols for high-throughput use .Selection agents: Transfection is used in conjunction with an appropriate selection agent, i .e ., Geneticin®, hygromycin, blasticidin, or Zeocin™ .Protein translocation: With Voyager™ tech nology, active fusion proteins can be translocated across the eukaryotic cell membrane into cells . Voyager™ technology is based on the properties of the herpes simplex viral protein VP22 . Using Voyager™ translocation, you can study pro-tein function in cells that are extremely difficult to transfect . Voyager™ translocation results in a 100% homogeneous population because all of the cells will contain the translocated protein . The translocated protein can be studied almost immediately after it enters the cell . Fusion proteins can be generated in eukaryotic cells or E. coli .

Table 1—Guidelines for selecting a transfection reagent.

Transfection application Recommended reagent

Most adherent cells Lipofectamine™ 2000 Reagent Lipofectamine™ LTX with PLUS™ Reagent

Animal origin–free Lipofectamine™ 2000 CD Reagent FreeStyle™ MAX Reagent

RNAi Lipofectamine™ 2000 Reagent Lipofectamine™ RNAiMAX Reagent

Oligonucleotides (antisense) Oligofectamine™ Reagent

Cells plated at low density Lipofectamine™ LTX with PLUS™ Reagent

Suspension cells FreeStyle™ MAX Reagent Lipofectamine™ LTX with PLUS™ Reagent

Suspension 293 cells FreeStyle™ MAX Reagent 293fectin™ Reagent

Suspension CHO cells FreeStyle™ MAX Reagent

Insect cells Cellfectin® Reagent

Endothelial cells Lipofectin™ LTX with PLUS™ Reagent

Difficult-to-transfect cells Lipofectamine™ LTX with PLUS™ Reagent

Primary cells Lipofectamine™ LTX with PLUS™ Reagent

Figure 1—Introduction of DNA into mammalian cells. For cell line–specific transfection protocols, visit the Cell Lines database at www .invitrogen .com/transfection .

Cationic lipid reagent

Mammalian cell Protein is produced

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++

+ +

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+

+ +

++

+++

+

+ ++

+

+

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+ +

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+

+ +

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+

+ ++

+

+

++

++

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+ +

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+

+ ++

+

+++

+

+ ++

++

++

++

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+ +

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+

+ +

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+++

+

+ ++

+

+

++

N

C

++

++

++++

+

++++

++

++

++ +++++

DNA

DNA–cationic lipid complex



4



Transfection Reagents

Lipofectamine™ 2000 Transfection ReagentLipofectamine™ 2000 Transfection Reagent is a proprietary cationic lipid formulation that offers the highest transfection efficiencies on the widest variety of cell lines with high protein expression levels or gene knockdown activity (RNAi) . It is the leading transfection reagent for simple, effective, and optimal nucleic acid delivery in a wide range of eukaryotic cell types and is proven to work in high-throughput applications .Recommended storage conditions: 2°C to 8°C .

Ordering InformationQuantity Cat. no.0 .75 ml 11668-027

1 .5 ml 11668-019

15 ml 11668-500


FREE Lipofectamine™ 2000 CD Transfection ReagentLipofectamine™ 2000 CD (Chemically Defined) Transfection Reagent is designed for applications where concerns about animal-derived components exist . It is synthesized from 100% animal origin–free components, making it easy to validate the absence of zoonotic dis-eases, such as BSE or viruses, in experiments or cell lines . Lipofectamine™ 2000 CD Reagent is appropriate for all transfections from preclinical or academic research to large-scale bioprocess applications .Recommended storage conditions: 2°C to 8°C . Notes: A Drug Master File (DMF) describing the manufacture of Lipofectamine™ 2000 CD has been filed with to the FDA . The DMF details the process to manufacture Lipofectamine™ 2000 CD using animal origin–free components, simplifying the FDA documentation required for your downstream product . Permission to cross-reference the Drug Master File may be obtained by contacting Invitrogen Technical Support or your local Sales Representative .


1 ml 12566-014

Related products PageOptiPRO™ SFM (1X) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .180


FREE Lipofectamine™ LTX Transfection ReagentLipofectamine™ LTX Reagent offers a new, advanced solution for gene expression studies in hard-to-transfect cells . No other plasmid DNA–specific transfection reagent can match the efficiency, convenience, and gentleness of Lipofectamine™ LTX Reagent for transfection of primary, difficult-to-transfect, and sensitive cell lines . Lipofectamine™ LTX Reagent is synthesized from 100% animal origin–free components, making it easy to validate the absence of zoonotic diseases, such as BSE or viruses, in experi-ments or cell lines . Lipofectamine™ LTX Reagent offers high transfection efficiency with maximal (>90%) cell viability using rapid, simple protocols that obviate the need for changing media after transfection .

PLUS™ reagent enhances transfection efficiency of Lipofectamine™ LTX reagent in primary and hard-to-transfect cells . Recommended storage conditions: 2°C to 8°C .


1 ml 15338-100

15 ml 15338-500


FREE FreeStyle™ MAX ReagentGenerating milligram quantities of functional protein in mammalian cells has been a big challenge to pharmaceutical and preclinical researchers . The new FreeStyle™ MAX Reagent is a proprietary cationic lipid formulation that allows rapid generation of milligram quantities of recombinant protein in suspension CHO and 293 cells . Combining transient transfection with suspension culture protocols reduces the time required to generate milligram levels of protein from several months to one week . Protein production in CHO cells produces proteins with glycosylation patterns required for many downstream preclinical and therapeutic applications . Recommended storage conditions: 2°C to 8°C .


1 ml 16447-100

15 ml 16447-500

10 x 15 ml 16447-750

Related products PageFreeStyle™ 293 Expression Medium . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .177FreeStyle™ 293 Expression System . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .178FreeStyle™ 293-F Cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .177


132


293fectin™ Transfection Reagent293fectin™ Transfection Reagent is a specially designed cationic lipid formulation that offers extremely high transfection efficiencies of FreeStyle™ 293-F suspension cells in serum-free Gibco® FreeStyle™ 293 Expression Medium . It is available separately or as part of the FreeStyle™ 293 Expression System .Recommended storage conditions: 2°C to 8°C .


1 ml 12347-019 15 ml 12347-500

10 x 15 ml 12347-750

Lipofectamine™ RNAiMAX Transfection ReagentLipofectamine™ RNAiMAX Transfection Reagent offers an advanced, efficient solution for siRNA delivery . No other siRNA-specific transfection reagent provides such easy and efficient siRNA delivery in a wide variety of cell lines . High knockdown levels of target genes can be achieved with as little as 1 nM siRNA . Lipofectamine™ RNAiMAX Transfection Reagent gives maximal knockdown and excellent cell viability across a 10-fold concentration range of the reagent . This makes Lipofectamine™ RNAiMAX reagent easy to optimize so results are easy to obtain in every experiment .


0 .75 ml 13778-075

1 .5 ml 13778-150

Cellfectin® II Transfection ReagentRecommended storage conditions: +4°C to 8°C .


1 ml 10362-010

PLUS™ ReagentPLUS™ Reagent was specifically designed to expand the array of transfectable cell types . Some customers find when using PLUS™ Reagent, less optimization of DNA concentration is needed to achieve high transfection performance . PLUS™ Reagent used with Lipofectamine™ LTX Reagent has been demonstrated to improve the transfection of BHK-21, NIH 3T3, and other cell lines .1

Recommended storage conditions: 2°C to 8°C .

Reference1 . Fawcett, T .W . and Hawley-Nelson, P . (1997) FOCUS® 19, 5 .


0 .85 ml 11514-015

Lipofectamine™ Transfection ReagentLipofectamine™ Reagent is formulated for the transfection of DNA into eukaryotic cells and has been shown to work well with cells such as BHK-21 and NIH 3T3 .Recommended storage conditions: 2°C to 8°C .


0 .5 ml 18324-111

1 ml 18324-012

4 ml 18324-020

Lipofectamine™ 2000 Transfection ReagentLipofectamine™ 2000 Reagent delivers DNA with excellent transfection performance for protein expression, gene silencing and functional assays . Superior efficiency, optimized protocols, and unparalleled success across a broad range of cell lines (figure 1) - Lipofectamine™ 2000 Reagent does it all .Recommended storage conditions: 2°C to 8°C .


0 .75 ml 11668-027

1 .5 ml 11668-019

15 ml 11668-500



4



Notes:


134


Notes:



4



Notes:


136



Chapter 5 Balanced Salt SolutionsBalanced Salt Solutions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 138

Dulbecco’s Phosphate-Buffered Salines . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 138

Dulbecco’s Phosphate-Buffered Saline (DPBS) (1X), liquid. . . . . . . . . . . . . . . . . . . . . . . . . . 138

Dulbecco’s Phosphate-Buffered Saline (DPBS) (10X), liquid . . . . . . . . . . . . . . . . . . . . . . . . . 139

Dulbecco’s Phosphate-Buffered Saline (DPBS), powder . . . . . . . . . . . . . . . . . . . . . . . . . . . 139

Earle’s Balanced Salt Solution. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 139

Earle’s Balanced Salt Solution (EBSS) (1X), liquid . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 139

Hanks’ Balanced Salt Solutions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 140

Hanks’ Balanced Salt Solution (HBSS) (1X), liquid . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 140

Hanks’ Balanced Salt Solution (HBSS) (10X), liquid. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 140

Phosphate-Buffered Saline (PBS) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 141

Phosphate-Buffered Saline (PBS) 7.2 (1X), liquid . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 141

Phosphate-Buffered Saline (PBS) 7.2 (10X), liquid . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 141

Phosphate-Buffered Saline (PBS) 7.4 (1X), liquid . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 141

Phosphate-Buffered Saline (PBS) 7.4 (10X), liquid . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 141

Balanced Salt Solutions Preparation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 142

Balanced Salt Solutions Formulations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 143

Chapter 5 Balanced Salt Solutions

138


Balanced Salt SolutionsFor information on classifications, production, or preparation of the products in this chapter, see page 142.

Liquid balanced salt solutionsQuality Control

To ensure that our validated systems and controls continue to be effective, we confirm the quality of our balanced salt solutions by testing samples from each production batch.Chemical checks. We measure osmolality and pH of all batches to verify compliance with product specifications.Microbiological tests. For balanced salt solutions, we confirm the absence of bacterial and fungal contamination using the method referenced in the current edition of the U.S. Pharmacopeia (USP) sterility test monograph.Stability tests. The expiration date shown on every in vitro diagnostic (IVD)-labeled product is an indication of how long the product will deliver effective performance. Our stability program establishes and verifies product expiration dates. Product groups are monitored at periodic intervals to determine the length of time that the product will deliver acceptable performance under recommended storage conditions.Endotoxin tests. We determine endotoxin levels in all balanced salt solutions using a Limulus Amoebocyte Lysate (LAL) test. Salt solutions with the lowest level of endotoxin attainable in the industry today are routinely available from Invitrogen.

Powdered balanced salt solutionsQuality Control

We confirm the quality of our powdered balanced salt solutions by evaluating samples from each batch as required by specifications. Samples are prepared in 1X liquid form and membrane filtered, and testing may include:1. Endotoxin2. Solubility3. Osmolality4. pHThe values for these parameters are determined using the test conditions described for liquid balanced salt solutions. All powdered balanced salt solutions are produced without sodium bicarbonate to increase stability.

Dulbecco’s Phosphate-Buffered SalinesAll gibco® Dulbecco’s Phosphate-Buffered Salines contain potassium chloride, monobasic potassium phosphate, sodium chloride, and dibasic sodium phosphate; some formulations may contain additional components.For product formulation please refer to the tables at the end of this chapter.

Dulbecco’s Phosphate-Buffered Saline (DPBS) (1X), liquidRecommended storage conditions: 15°C to 30°C.Intended use: In vitro diagnostic (IVD).


100 ml 14040-083

500 ml 14040-091

10 × 500 ml (case) 14040-174

1,000 ml 14040-117

6 × 1,000 ml (case) 14040-224

Contains calcium and magnesium, but no phenol red.

Quantity Cat. no.

500 ml 14190-094

10 × 500 ml (case) 14190-169

1,000 ml 14190-136

6 × 1,000 ml (case) 14190-326

5L 14190-342

10L 14190-359

20L 14190-367

Contains no calcium, magnesium, or phenol red.

Recommended storage conditions: 15°C to 30°C.Quantity Cat. no.

500 ml 14287-080

1,000 ml 14287-072

Contains 1,000 mg/L D-glucose and 36 mg/L sodium pyruvate, calcium, and mag-nesium, but no phenol red.

gibco® cell culture catalogueBalanced Salt Solutions

5



Comparison of Dulbecco’s Phosphate-Buffered Salines (DPBS)Base cat. no. 14040* 14287 14080 21300 14190 14200 21600

With 1,000 mg/L D-glucose and 36 mg/L sodium pyruvate ●

Without calcium chloride and magnesium chloride ● ● ● ●

Without phenol red ● ● ● ● ● ● ●

10X concentration ● ●

Powder † ● ●

* Standard formulation, as published in (1954) J. Exp. Med. 99, 167.

† Powdered formulations of DPBS, CaCl2 is supplied separately at 0.1 g/L.


For the exact composition of each product, see the formulation tables on page 115.

Earle’s Balanced Salt SolutionFor product formulation please refer to the tables at the end of this chapter.

Earle’s Balanced Salt Solution (EBSS) (1X), liquidRecommended storage conditions: 15°C to 30°C.Intended use: In vitro diagnostic (IVD).


500 ml 14155-048

Contains no calcium, magnesium, or phenol red.

Dulbecco’s Phosphate-Buffered Saline (DPBS) (10X), liquidpH: The pH of the 10X solution is 4.4 to 4.7. The pH of the 1X solution will be acidic after 1:10 dilution and must be adjusted to 7.1 ± 0.1.Recommended storage conditions: 15°C to 30°C.Intended use: In vitro diagnostic (IVD).


500 ml 14080-048


pH: The pH of the 10X solution is 6.7 to 7.0. The pH of the 1X solution should be checked after 1:10 dilution to ensure that it is 7.1 ± 0.1. If the pH is not 7.1 ± 0.1, adjust as outlined in Balanced Salt Solutions Preparation, page 123.

Quantity Cat. no.

500 ml 14200-067

10 × 500 ml (case) 14200-083

Contains no calcium or magnesium.

Dulbecco’s Phosphate-Buffered Saline (DPBS), powderRecommended storage conditions: 15°C to 30°C.Intended use: In vitro diagnostic (IVD).


1 × 10 L 21600-069

10 × 1 L 21600-010

1 × 50 L 21600-044

Contains no calcium or magnesium.

Quantity Cat. no.

1 × 10 L 21300-058

1 × 1 L 21300-017

Contains magnesium. Calcium chloride is supplied separately in an amount suf-ficient for 0.1 g/L.


140


Hanks’ Balanced Salt SolutionsAll gibco® Hanks’ Balanced Salt Solutions contain potassium chloride, mono basic potassium phosphate, sodium chloride, dibasic sodium phos phate, and D-glucose; some formulations may contain additional components.For product formulation please refer to the tables at the end of this chapter.

Comparison of Hanks’ Balanced Salt Solutions (HBSS)Base cat. no. 24020* 14025 14065 14170 14175* 14185

Without calcium chloride, magnesium chloride, or magnesium sulfate ● ● ●

Without phenol red ● ● ● ● ●

10X concentration † ● ●

* Standard formulation, as published in Hanks, J.H. and Wallace, R.E. (1949) Proc. Soc. Exp. Biol. Med. 71, 196, Modification—National Institutes of Health. † MgCl2•6H2O added to original formulation. Original formulation calls for 0.06 g/L Na2PO4•2H2O and 0.02 g/L phenol red. Hanks’ Balanced Salt Solutions are intended for use in ambient air, not in CO2-equilibrated incubators.


For the exact composition of each product, see the formulation tables on page 127.

Hanks’ Balanced Salt Solution (HBSS) (1X), liquidRecommended storage conditions: 15°C to 30°C.Intended use: In vitro diagnostic (IVD).


500 ml 24020-091

Contains calcium and magnesium.

Quantity Cat. no.

500 ml 14025-050

1,000 ml 14025-076

10 × 500 ml (case) 14025-100

6 × 1,000 ml (case) 14025-126


Quantity Cat. no.

100 ml 14170-070

500 ml 14170-088

10 × 500 ml (case) 14170-138

5L 14170-195

10L 14170-203

Contains no calcium chloride, magnesium chloride, or magnesium sulfate.

Quantity Cat. no.

500 ml 14175-053

10 × 500 ml (case) 14175-129

1,000 ml 14175-079

6 × 1,000 ml (case) 14175-145

Contains no calcium chloride, magnesium chloride, magnesium sulfate, or phenol red.

Hanks’ Balanced Salt Solution (HBSS) (10X), liquidRecommended storage conditions: 15°C to 30°C.Intended use: In vitro diagnostic (IVD).


500 ml 14185-045

Contains no calcium chloride, magnesium chloride, magnesium sulfate, sodium bicarbonate, or phenol red.

Quantity Cat. no.

500 ml 14065-049

Contains calcium and magnesium, but no sodium bicarbonate or phenol red.


5



Phosphate-Buffered Saline (PBS)All GIBCO® Phosphate-Buffered Salines contain monobasic potassium phosphate, sodium chloride, and dibasic sodium phosphate; some formu lations may contain additional components. For the exact composition of each product, see the formulation tables on page 144.For product formulation please refer to the tables at the end of this chapter.

Phosphate-Buffered Saline (PBS) 7.2 (1X), liquidpH: 7.2 ± 0.1Intended use: In vitro diagnostic (IVD).


500 ml 20012-019

10 × 500 ml (case) 20012-068

1,000 ml 20012-043

Phosphate-Buffered Saline (PBS) 7.2 (10X), liquidpH: The pH of the 10X solution is 7.1 to 7.3. The pH may change upon dilution to 1X and require readjustment.Intended use: In vitro diagnostic (IVD).


500 ml 70013-016

10 × 500 ml (case) 70013-065

Phosphate-Buffered Saline (PBS) 7.4 (1X), liquidpH: 7.4 ± 0.1Intended use: In vitro diagnostic (IVD).


500 ml 10010-015

10 × 500 ml (case) 10010-056

1,000 ml 10010-031

6 × 1,000 ml (case) 10010-072

Phosphate-Buffered Saline (PBS) 7.4 (10X), liquidpH: The pH of the 10X solution is 7.3 to 7.5. The pH may change upon dilution to 1X and require readjustment.Intended use: In vitro diagnostic (IVD).


500 ml 70011-036

10 × 500 ml (case) 70011-051


142


Balanced Salt Solutions PreparationPreparing 1X solutions from10X concentratesTo prepare an acceptable final, single-strength solution, perform the following procedure under aseptic conditions. We recommend Distilled Water (base cat. no. 15230) and 7.5% Sodium Bicarbonate Solution (base cat. no. 25080) for use in this protocol. Use sterile solutions of 1 N sodium hydroxide or hydrochloric acid prepared by membrane filtration using appropriate filters. Also note that, because we adjust the pH of all 10X balanced salt solution concentrates for solubility, you may need to adjust the pH after dilution and add sodium bicarbonate, as appropriate.1. Aseptically dilute 100 ml of 10X concentrate with approximately

850 ml of sterile distilled water.2. Aseptically add the correct amount of 7.5% sodium bicarbonate solu-

tion. See the table below for concentrations.3. Adjust the pH as necessary with 1 N hydrochloric acid or 1 N sodium

hydroxide. See the table below for 1X pH values.4. QS to volume with sterile distilled water.5. Dispense the solution into sterile containers. Cap the bottles

tightly with sterile closures and store them at the recommended temperature.

Preparing powdered Dulbecco’s Phosphate-Buffered Saline (DPBS)1. To a mixing container that is as close to the final volume as possible,

add 2–3% less distilled water than the desired total volume of DPBS.2. Add powdered DPBS (less calcium chloride) to room temperature

(20°C to 30°C) water with gentle stirring. Do not heat the water.3. Rinse the inside of the package to remove all traces of powder. Be sure

all material goes into solution before proceeding to the next step.4. Add 0.1 g of calcium chloride per liter of DPBS.5. Dilute the DPBS to the desired volume with water and stir until

dissolved. Do not overmix.6. Process the DPBS immediately into sterile containers by membrane

filtration using a 0.2 µm filter. We recommend using a positive-pressure system.

Sodium Bicarbonate Concentrations Achieved Using 7.5% Sodium Bicarbonate Solution (base cat. no. 25080)NaHCO3

pH of 10X solution Final concentration (g/L) 7.5% solution to add (ml/L)

pH range after 1:10 dilution and addition of NaHCO3 Recommended pH of 1X solution*

Base cat. no. Balanced salt solutions

14065 5.8–6.1 0.35 4.7 7.5–7.8 7.0–7.4

14080 † 4.4–4.7 – – 5.2–5.5 7.0–7.2

14185 5.8–6.1 0.35 4.7 6.0–6.3 7.0–7.4

14200 † 6.7–7.0 – – 7.1–7.4 7.0–7.2

* pH range will vary depending on pH of water used for dilution. † Formulation does not require addition of NaHCO3.


5



Balanced Salt Solutions Formulations

Dulbecco’s Phosphate-Buffered Saline (DPBS)1

Base cat. no. 14040 14080 14190 14300 14287 21300 21600

Component 1X liquid, g/L 10X liquid, g/L 1X liquid, g/L 10X liquid, g/L 1X liquid, g/L Powder, g/L Powder, g/L

Inorganic salts

CaCl2 (anhydrous) 0.10 1.00 – – 0.10 * –

KCl 0.20 2.00 0.20 2.00 0.20 0.20 0.20

KH2PO4 0.20 2.00 0.20 2.00 0.20 0.20 0.20

MgCl2 (anhydrous) – – – – – 0.047 –

MgCl2•6H2O 0.10 1.00 – – 0.10 – –

NaCl 8.00 80.00 8.00 80.00 8.00 8.00 8.00

Na2HPO4 – – – – – 1.15 1.15

Na2HPO4•7H2O 2.16 21.60 2.16 21.60 2.16 – –

Phenol red – – – – – – –

D-Glucose – – – – 1.00 – –

Sodium pyruvate – – – – 0.036 – –

* Supplied separately at 0.1 g/L. 1. J. Exp. Med. (1954) 98, 167.

Hanks’ Balanced Salt Solutions (HBSS)1

Base cat. no. 14025 14065 14170 14175 14185 24020

Component 1X liquid, g/L 10X liquid, g/L 1X liquid, g/L 1X liquid, g/L 10X liquid, g/L 1X liquid, g/L

Inorganic salts

CaCl2 (anhydrous) 0.14 1.40 – – – 0.14

KCl 0.40 4.00 0.40 0.40 4.00 0.40

KH2PO4 0.06 0.60 0.06 0.06 0.60 0.06

MgCl2•6H2O* 0.10 1.00 – – – 0.10

MgSO4•7H2O 0.10 1.00 – – – 0.10

NaCl 8.00 80.00 8.00 8.00 80.00 8.00

NaHCO3 0.35 – 0.35 0.35 – 0.35

Na2HPO4 0.048 – 0.048 0.048 – 0.048

Na2HPO4•7H2O † – 0.90 – – 0.90 –

Other components

D-Glucose 1.00 10.00 1.00 1.00 10.00 1.00

Phenol red ‡ – – 0.01 – – 0.01

* MgCl2•6H2O added to original formulation. † Original formulation calls for 0.06 g/L Na2HPO4•2H2O. Proc. Soc. Exp. Biol. Med. (1949) 71, 196. ‡ Original formulation calls for 0.02 g/L. Proc. Soc. Exp. Biol. Med. (1949) 71, 196.1. Hanks, J.H., and Wallace, R.E. (1949) Proc. Soc. Exp. Biol. Med. 71, 196, Modification—National Institutes of Health.


144


Phosphate-Buffered Saline (PBS)Base cat. no. 10010 20012 70011 70013

Component 1X liquid, g/L 1X liquid, g/L 10X liquid, g/L 10X liquid, g/L

Inorganic salts

KH2PO4 0.144 0.21 1.44 2.10

NaCl 9.00 9.00 90.00 90.00

Na2HPO4•7H2O 0.795 0.726 7.95 7.26

Earle’s Balanced Salt Solutions (EBSS)1

Base cat. no. 14155

Component 1X liquid, g/L

Inorganic salts

KCl 0.40

NaCl 6.80

NaHCO3 2.20

NaH2PO4•H2O* 0.14

Other components

D-Glucose 1.00

* Original formulation calls for 150.0 mg/L NaH2PO4•2H2O. In Vitro (1974) 9:6.1. J. Nat. Cancer Inst. (1943) 4, 167.


5



Notes:


146


Notes:


Cell Culture Reagents . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 148

new ACK Lysing Buffer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 149

Amino Acids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 149

GlutaMAX™-I Supplement . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 149

L-Glutamine-200 mM, liquid . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 149

L-Glutamine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 149

MEM Amino Acids Solution (50X), liquid . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 149

MEM Non-essential Amino Acids Solution . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 149

Antibiotics and Antimycotics. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 150

Actinomycin D . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 150

Ampicillin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 150

Antibiotic-Antimycotic . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 150

Blasticidin S HCl . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 151

Puromycin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 151

Carbenicillin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 151

Geneticin® Selective Antibiotic . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 152

Gentamicin Reagent . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 153

Hygromycin B . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 153

Kanamycin Sulfate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 153

Mycophenolic Acid . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 154

Neomycin Sulfate. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 154

Penicillin-Streptomycin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 154

Penicillin-Streptomycin-Glutamine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 154

Penicillin-Streptomycin-Neomycin (PSN) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 155

Polymyxin B Sulfate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 155

Streptomycin Sulfate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 155

Zeocin™ . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 155

Antifoaming and Defoaming Reagents . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 156

Biological Buffers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 157

Cell Dissociation Products. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 158

Distilled Water . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 161

new WFI for Cell Culture (Water for Injection) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 161

Attachment Factors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 162

Growth Supplements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 163

Hybridoma Reagents . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 166

Insect Medium Supplements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 167

Mycoplasma Detection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 167

Other Cell Culture Reagents . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 168

Cell Culture Reagent Formulations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 169

new Presto Blue™. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 170

Chapter 6 Standard Cell Culture Reagents


148


Cell Culture ReagentsQuality ControlFor specific information on quality control, please refer to the individual product descriptions. Each production batch is analyzed as required by specifications. Testing may include:Chemical checks. Osmolality and pH are tested to verify that batches meet product specifications.Microbiological tests. Samples of liquid and lyophilized products are tested to confirm the absence of bacterial and fungil contamination using a protocol or the method referenced in the current edition of the U.S. Pharmacopeia sterility test monograph. We also test products of animal origin for the absence of mycoplasmas using the large-volume inoculation method of Barile and Kern 1 and/or the DNA staining method of Chen.2 For details of these tests, see Chapter 5, Animal Sera, “Microbiological Tests.”Stability testing program. The expiration date shown on every in vitro diagnostic (IVD)-labeled product is an indication of how long the product will deliver effective performance. Our stability testing program establishes and verifies product expiration dates. Product groups are monitored at periodic intervals to determine the length of time that the product will deliver acceptable performance under recommended storage conditions.Functional growth tests. Products are tested for functionality, growth promotion, and cytotoxicity as appropriate.Attention: Researchers using serum-free or reduced-serum conditions or other sensitive cell culture systems should determine cytotoxicity for their specific applications.

References1. Barile, M.F. and Kern, J. (1971) Proc. Soc. Exp. Biol. Med. 138, 432.2. Chen, T.R. (1977) Exp. Cell Res. 104, 255.

Custom Products and PackagingMany of the dry chemicals used in the manufacture of our products are available in bulk. While they may not be listed in this chapter, please contact our Custom Products Services goup for availability, minimum order, and minimum packagingr.

Biochemicals are specified as USP, ACS, FCC, or NF grade as applicable. If no referenced specifications exist, standards are established by Invi trogen. Material Safety Data Sheets or Certificates of Analysis are available upon request. All of these products are intended for use by qualified professionals who will safely handle and dispose of them.

gibco® cell culture catalogue6

Standard Cell Culture Reagents



ACK Lysing Buffer

new ACK Lysing BufferACK Lysing Buffer is suitable for use in the lysis of red blood cells in EDTA-treated whole-blood samples.

One volume of ACK Lysing Buffer is sufficient to lyse 10–20 times the volume of the blood sample.Recommended storage conditions: 15°C to 30°C.


100 ml A1049201

Amino AcidsGlutaMAX™-I SupplementGlutaMAX™-I Supplement can be used as a direct substitute for L-glutamine for most cell types. GlutaMAX™-I Supplement is only removed from the medium by cell metabolism. There is no build-up of toxic metabolites by spontaneous breakdown.

200 mM supplied in 0.85% NaCl. ■

Extremely stable in aqueous solution. See Appendix I, page ■ 202.Recommended storage conditions: –5°C to –20°C.

Reference1. Christie, A. and Butler, M. (1994) FOCUS® 16, 9.


100 ml 35050-038

20 × 100 ml (case) 35050-087

Contains the dipeptide L-alanyl-L-glutamine.

L-Glutamine-200 mM, liquidSupplied at 29.2 mg/ml in 0.85% NaCl.pH: 4.7 to 6.0.Recommended storage conditions: –5°C to –20°C.Intended use: In vitro diagnostic (IVD).


20 ml 25030-032

20 × 100 ml (case) 25030-123

L-GlutamineUSP Grade.C5H10N2O3

Formula weight: 146.Recommended storage conditions: 15°C to 30°C.


1 kg 21051-040

MEM Amino Acids Solution (50X), liquidPrepared in distilled water. The amino acids in this solution are 50X the concentration in a standard MEM Medium.For product formulation please refer to the tables at the end of this chapter.pH: 0.6 to 1.2.Recommended storage conditions: 2°C to 8°C.Intended use: In vitro diagnostic (IVD).Attention: This material is hazardous. All persons using this product should review the Material Safety Data Sheet before handling.


100 ml 11130-036

20 x 100ml 11130-077


MEM Non-essential Amino Acids Solution 10 mM (100X), liquidPrepared in distilled water. The non-essential amino acids in this solution are 100X the concentration in a MEM-α Medium.For product formulation please refer to the tables at the end of this chapter.pH: 0.6 to 1.7.Recommended storage conditions: 2°C to 8°C.Intended use: In vitro diagnostic (IVD).Attention: This material is hazardous. All persons using this product should review the Material Safety Data Sheet before handling.


100 ml 11140-035

20 × 100 ml (case) 11140-068


150


Antibiotics and AntimycoticsWe supply a wide range of antibiotic mixtures that are useful for the prophylactic control or elimination of a broad spectrum of microbial contaminants from mammalian cell cultures. Some of these antibiotics may also be used in the selection of resistant bacteria. Gibco® anti biotic mixtures are subjected to carefully controlled manufacturing procedures to ensure that you always receive products of consistently high quality.Quality control of raw materials. All antibiotics selected for man ufacture of gibco® antibiotic mixtures are received from approved suppliers. Antibiotic mixtures are formulated according to the product potency as determined by an approved independent testing laboratory.Final product quality control. Our antibiotic mixtures are tested for the absence of bacteria and fungi, utilizing protocols or those referenced in the USP. The pH and osmolality levels must be within established specifications.Use of antibiotics in cell culture systems. The dilutions indicated in the product listing for each antibiotic mixture usually are sufficient to control a broad spectrum of microbial contaminants in a wide range of mammalian cell types. However, prior to using these anti biotic mixtures, we caution users to be aware of the effects that these dilutions could have on specific cell culture systems. Prescreening potent additives is a recognized quality assurance procedure. Periodic discontinuation of antibiotic use is recommended for several reasons: questionable cell culture techniques can be masked, antibiotic-resistant organisms may arise, and cryptic microbial contaminants may be present and be masked by antibiotic use.

Actinomycin DC62H86N12O16 (nonsterile)Formula weight: 1,255 (anhydrous).Recommended storage conditions: 2°C to 8°C.Attention: This material is hazardous. All persons using this product should review the Material Safety Data Sheet before handling.


5 mg 11805-017

Ampicillin, Sodium Salt, lyophilized200 mg of ampicillin sodium salt per vial.Applications: Selection of resistant bacteria.Recommended storage conditions: 2°C to 8°C before rehydration; –5°C to –20°C after rehydration.Attention: This material is hazardous. All persons using this product should review the Material Safety Data Sheet before handling.


20 ml 11593-027

References1. Miller, J.H. (1972) Experiments in Molecular Genetics, p. 433, Cold Spring Harbor

Laboratory, Cold Spring Harbor, New York.2. Davis, R.W., Botstein, D., and Roth, J.R. (1980) Advanced Bacterial Genetics, p. 204,

Cold Spring Harbor Laboratory, Cold Spring Harbor, New York.

Antibiotic-Antimycotic (100X), liquidSpectrum: Bacteria, fungi, and yeasts.Recommended storage conditions: –5°C to –20°C.Attention: This material is hazardous. All persons using this product should review the Material Safety Data Sheet before handling.

Fungizone™ is a trademark of E.R. Squibb & Sons, LLC.


20 ml 15240-096

100 ml 15240-062

Contains 10,000 units of penicillin (base), 10,000 µg of streptomycin (base), and 25 µg of amphotericin B per ml, utilizing penicillin G (sodium salt), streptomycin sulfate, and amphotericin B as Fungizone™ Anti my cotic in 0.85% saline.





Blasticidin S HClC17H26N8O5•HClFormula weight: 458.9.Blasticidin S is a nucleoside selection agent isolated from Streptomyces griseochromogenes. It is a potent translational inhibitor in both pro karyotic and eukaryotic cells.1 E. coli strains are generally sensitive to 50 µg/ml while mammalian cells are sensitive to concentrations from 2 to 10 µg/ml. Cell death occurs rapidly at low concentrations. Resis tance to blasticidin is conferred by the products of the bsd gene from Aspergillus terreus.2 Blasticidin-resistant mammalian cell lines can be generated in as little as seven days post-transfection.Recommended storage conditions: Blasticidin S HCl is provided as a powder and should be stored at –20°C. Blasticidin is guaranteed stable for 6 months when properly stored.

Blasticidin and the blasticidin resistance gene (bsd) are the subject of US Patent No. 5,527,701 sold under patent license for research purposes only. Inquiries for commercial use should be directed to: Kaken Pharmaceutical Company, Ltd., Bunkyo Green Court, Center Office Building, 19-20 Fl, 28-8 Honkomagome 2-chome, Bunkyo-ku, Tokyo 113-8650, Japan, Tel: + 81 3-5977-5008; Fax: + 81 3-5977-5008.


50 mg R210-01

20ml A1113-902

10 x 1ml A1113-903

PuromycinC17H26N8O5•HCl C22H29N7O5, 2HClFormula weight: 458.9.Puromycin Dihydrochloride is an aminonucleoside antibiotic produced by Streptomyces alboniger. It inhibits peptidyl transfer on both prokaryotic and eukaryotic ribosomes. This antibiotic also inhibits the growth of Gram positive bacteria and Morevarious animal and insect cells. Resistance to Puromycin is conferred by the expression of the “pac” gene. It is widely used in cell biology as selective agent in mammalian and stem cell culture systems.Format: Liquid Solution (Sterile Filtered), 10mg⁄mLRecommended storage conditions: Product shipped in a box containing 10 x 1ml vials for single use, storage -20 deg C.


20ml A1113-802

10 x 1ml A1113-803

Carbenicillin, Disodium SaltC17H26N8O5•HClFormula weight: 458.9.Application: Used in plant transformation for the killing of Agro bac terium following co-cultivation of the bacteria with plant tissue.1 Recommended storage conditions: 2°C to 8°C.

Reference1. Lin, J.J., Assad-Garcia, N., and Kuo, J. (1994) FOCUS® 16, 72.


5 g 10177-012


152


Geneticin® Selective Antibiotic.

Geneticin® Selective Antibiotic, liquid50 mg/ml active Geneticin® Selective Antibiotic in distilled water. Aseptically filtered.Applications: Geneticin® Selective Antibiotic, an aminoglycoside related to gentamicin,1–3 is used as a selective antibiotic in molecular genetics experiments.4,5 It is toxic to bacteria,1,2 yeast,3,4 higher plant 5 and mammalian cells,6–8 and protozoans and helminths.9,10 The resistance genes are dominant and are located on both transposons Tn601(903) and Tn5. The gene on Tn5 is referred to as aminoglycoside phosphotransferase 3´(II) or APH(3´)II, whereas the gene on Tn601 is referred to as aminoglycoside phosphotransferase 3´(I) or APH(3´)I. The genes are bacterial in derivation, but they can be expressed in eukaryotic cells. Intro duction of either of these genes into cells can confer resistance to Geneticin® Selective Antibiotic, which enables the cells to grow in media containing Geneticin® Selective Antibiotic.Properties:

Specific rotation: 104°C to 121°C on dried basis in waterFormula: C20H40O10N4•2H2SO4

Molecular weight: 496.5 (base), 692.7 (sulfate)Potency: ≥700 µg/mgElemental analysis: C = 34.68, N = 8.09, H = 6.40 (sulfate)Heavy metals: <20 ppm as Pb

Stability: Stable up to 24 months at –20°C or at 2°C to 8°C.Notes: The amount of Geneticin® Selective Antibiotic required in culture media to select for resistant cells varies. For a multiplying cell, 3 to 7 days will be required for death to occur. See the table for guidelines. For a discussion of performance parameters, such as potency and working range, please visit www.invitrogen.com/geneticin.Recommended storage conditions: –20°C, or at 2°C to 8°C.Attention: This material is hazardous. All persons using this product should review the Material Safety Data Sheet before handling.

Geneticin® Selective Antibiotic: Guide to Concentration

Cells Transposon Gene Concentration (mg/L) Notes References

Dictyostelium Tn5 APH(3´)-II 10* 35*

Cells grown in medium Cells plated on lyophilized bacteria 8

Mammalian Tn5 APH(3´)-II 400* 200*

For selection For maintenance 6, 7

Plant Tn5 APH(3´)-II 10* For selection 5

Yeast Tn601 APH(3´)-I 500 125

For selection For maintenance 3

*Active drug

NH2

CH3

NH2

NH2

OH

2H2SO4

HO C–H

OH

O

O

O

HO

HO

NHCH3

H3C

OH

O

References1. Weinstein, M.J., Wagman, G.H., Testa, R.T., and Marquez, J.A. (May 25, 1976) Antibiotic G-418 and the Production Thereof, US Patent 3,959,254.2. Weinstein, M.J., Wagman, G.H., Testa, R.T., and Marquez, J.A. (December 14, 1976) Antibiotic G-418 and the Production Thereof, US Patent 3,997,403.3. Jiménez, A. and Davies, J. (1980) Nature 287, 869. 4. Davies, J. and Jiménez, A. (1980) Am. J. Trop. Med. Hyg. 29(5) Suppl: 1089.5. Ursic, D., Kemp, J.D., and Helgeson, J.P. (1981) Biochem. Biophys. Res. Comm. 101(3), 1031.6. Southern, P.J. (1981) In Vitro 18, 315.7. Southern, P.J. and Berg, P. (1982) J. Mol. Appl. Genet. 1, 327.8. Canaani, D. and Berg, P. (1982) Proc. Natl. Acad. Sci. USA 79, 5166. 9. Loebenberg, D., Counelis, M., and Waitz, J.A. (1975) Antimicrob. Agents Chemother. 7:6, 811.10. Waitz, J.A., Sabatelli, F., Menzel, F., and Moss, E.L., Jr. (1974) Antimicrob. Agents Chemother. 6:5, 579.


20 ml 10131-035

100 ml 10131-027





Geneticin® Selective Antibiotic, powderCrystalline, nonsterile (G418 sulfate)Stability: Very stable, may be stored in powdered form at room temperature (15°C to 30°C) for up to 3 years; soluble in water.Recommended storage conditions: 15°C to 30°C before reconstitution;2°C to 8°C after reconstitution.Attention: This material is hazardous. All persons using this product should review the Material Safety Data Sheet before handling.


1 g 11811-023

5 g 11811-031

10g 11811-064

25 g 11811-098

Gentamicin Reagent Solution, liquidSpectrum: Gram-positive and gram-negative bacteria.Recommended concentration: 0.5 to 50 µg/ml.Recommended storage conditions: 15°C to 30°C.Attention: This material is hazardous. All persons using this product should review the Material Safety Data Sheet before handling.

Ordering InformationSize Quantity Cat. no.

10 mg 100 ml 15710-049

20 × 10 ml (case) 15710-080

50 mg 100 ml 15750-045 20 × 10 ml (case) 15750-102

Contains 50 mg/ml gentamicin sulfate in distilled water.

Hygromycin B, liquidC20H37N3O13

Purity: >80%.Formula weight: 527.5.Assay: ≥50 mg/ml.Application: Used in plant transformation 1,2 and mammalian transfection systems 3-5 for the selection of resistant cells.Recommended storage conditions: 2°C to 8°C.

References1. Lin, J.J., Ma, J., Assad-Garcia, N., and Kuo, J. (1996) FOCUS® 18, 47.2. Datta, S.K., Peterhans, A., Datta, K., and Potrykus, I. (1990) Biotech. 8, 736.3. Gonzales, A., Jiménez, A., Vazquez, D., Davies, J.E., and Schindler, D. (1978)

Biochem. Biophys. Acta 521, 459.4. Blochlinger, K. and Diggelmann, H. (1984) Mol. Cell Biol. 4, 2929.5. Giordano, T.J. and McAllister, W.T. (1990) Gene 88, 285.


20 ml 10687-010

Kanamycin SulfateUSP gradeC18H36N4O11•H2SO4 (nonsterile)Molecular weight: 583Assay: ≥750 µg kanamycin/mg (anhydrous)Recommended storage conditions: 15°C to 30°C.Attention: This material is hazardous. All persons using this product should review the Material Safety Data Sheet before handling.


5 g 11815-024

25 g 11815-032

Kanamycin Sulfate (100X), liquidSpectrum: Gram-positive and gram-negative bacteria. Recommended storage conditions: –5°C to –20°C.Attention: This material is hazardous. All persons using this product should review the Material Safety Data Sheet before handling.


100 ml 15160-047

20 x 100 ml 15160-070

Contains 10 mg/ml kanamycin (base) utilizing kanamycin sulfate in 0.85% saline.


154


Mycophenolic AcidCrystalline, nonsterileApplications: Mycophenolic Acid (MPA) is an antibiotic that blocks inosine monophosphate dehydrogenase, an enzyme in the guanosine mono phosphate (GMP) synthesis pathway. This product can be used as a selective agent in mammalian systems because of the different effects of the activities of mammalian HPRT (hypoxanthine phosphoribosyl transferase) and bacterial XGPRT (xanthine guanine phosphoribosyltrans-ferase). Both enzymes mediate an alternative (salvage) pathway for GMP synthesis. Because mammalian cells can use xanthine to a limited extent during GMP synthesis, GMP synthesis is effectively blocked by MPA in guanine-free media. Addition of Aminopterin to the medium has been found to enhance this blockage. XGPRT of E. coli can effectively convert xanthine to xanthine monophosphate. The insertion of the Ecogpt gene, which codes for XGPRT, into mammalian cells allows these cells to grow in media containing 25 µg/ml of MPA. For more information, see the references below.Properties:

Formula: C17H20O6

Molecular weight: 320.35Purity: ≥95%Solubility: moderately soluble in ether and chloroform, freely soluble

in alcohol, insoluble in waterRecommended storage conditions: 15°C to 30°C before reconstitution; –5°C to –20°C after reconstitution.Attention: This material is hazardous. All persons using this product should review the Material Safety Data Sheet before handling.

References1. Cass, C.E., Lowe, J.K., Manchak, J.M., and Henderson, J.F. (1977) Cancer Res. 37,

3314.2. Lowe, J.K., Brox, L., and Henderson, J.F. (1977) Cancer Res.736.3. Mulligan, R.C. and Berg, P. (1981) Proc. Natl. Acad. Sci. USA 78, 2072.4. Gorman, C., Padmanabhan, R., and Howard, B.H. (1983) Science 221, 551.


500 mg 11814-019

Neomycin SulfateUSP grade, nonsterileToxicity data: ROTECS QP4375000.Recommended storage conditions: 15°C to 30°C.Attention: This material is hazardous. All persons using this product should review the Material Safety Data Sheet before handling.


100 g 21810-031

Penicillin-Streptomycin, liquidSpectrum: Gram-positive and gram-negative bacteria.Recommended concentration: 50–100 U of penicillin, 50–100 µg of streptomycin.Recommended storage conditions: –5°C to –20°C.Attention: This material is hazardous. All persons using this product should review the Material Safety Data Sheet before handling.


100 ml 15070-063

Contains 5,000 units of penicillin (base) and 5,000 µg of streptomycin (base) per ml utilizing penicillin G (sodium salt) and streptomycin sulfate in 0.85% saline.

Quantity Cat. no.

20 ml 15140-148

100 ml 15140-122

20 × 100 ml (case) 15140-130

Contains 10,000 units of penicillin (base) and 10,000 µg of streptomycin (base) per ml utilizing penicillin G (sodium salt) and streptomycin sulfate in 0.85% saline.

Penicillin-Streptomycin-Glutamine (100X), liquidUtilizes penicillin G (sodium salt) and streptomycin sulfate.Spectrum: Gram-positive and gram-negative bacteria.Recommended storage conditions: –5°C to –20°C.Attention: This material is hazardous. All persons using this product should review the Material Safety Data Sheet before handling.


100 ml 10378-016

Contains 10,000 units of penicillin (base), 10,000 µg of streptomycin (base), and 29.2 mg of L-glutamine per ml in 0.85% saline, in a 10 mM citrate buffer to maintain penicillin potency.





Penicillin-Streptomycin-Neomycin (PSN) Antibiotic Mixture (100X), liquidSpectrum: Gram-positive and gram-negative bacteria.Recommended storage conditions: –5°C to –20°C.Attention: This material is hazardous. All persons using this product should review the Material Safety Data Sheet before handling.


100 ml 15640-055

Contains 5 mg of penicillin, 5 mg of streptomycin, and 10 mg of neomycin per ml utilizing penicillin G (sodium salt), streptomycin sulfate, and neomycin sulfate in 0.85% saline.

Polymyxin B SulfateUSP grade, nonsterile, crystallineSpectrum: Gram-negative bacteria.Recommended storage conditions: 15°C to 30°C.Attention: This material is hazardous. All persons using this product should review the Material Safety Data Sheet before handling.


25 MU 21850-029

Streptomycin SulfateUSP grade(C21H39N7O12)2•3H2SO4 (nonsterile)Formula weight: 1,457.Assay: 650 to 850 µg/mg (anhydrous).Application: Selection of resistant bacteria. Spectrum: Gram-negative bacteria. Recommended storage conditions: 2°C to 8°C.Attention: This material is hazardous. All persons using this product should review the Material Safety Data Sheet before handling


100 g 11860-038

Zeocin™C55H83N19O21S2CuFormula weight: 1137.41.Zeocin™ is an antibiotic in the bleomycin family. It causes cell death by intercalating into the DNA and cleaving it. Resistance to Zeocin™ is conferred by the Sh ble gene, which encodes a 13,665 DA protein. This protein confers resistance to Zeocin™ by binding to the antibiotic and preventing it from binding DNA. Zeocin™ is effective on most aerobic cells and can be used for selection in mammalian cell lines, yeast, and bacteria. The amount of Zeocin™ required for selection will vary with each cell type.Recommended storage conditions: Zeocin™ is provided in solution at a concentration of 100 mg/ml. Store Zeocin™ at –20°C in the dark. The drug is guaranteed stable for 6 months when properly stored.

Zeocin™ is a trademark of CAYLA.

The ble resistance gene is the subject of one or more of US Patent Nos. 5,021,344 and 5,118,620 and foreign equivalents owned by Cayla and licensed to Invitrogen. This product is sold for research purposes only. For commercial license information, please contact: Licensing Depart ment, Invitrogen Corporation, 5791 Van Allen Way, Carlsbad, CA 92008. Phone: 760-603-7200, Fax: 760-602-6500.


1 g R250-01

5 g R250-05


156


Antifoaming and Defoaming ReagentsFoamAway™ Irradiated Antifoaming/defoaming reagent.

Packaged in a convenient and versatile media bag that can be ■

coupled to your bioreactor via a sterile connection.Can be used with both mammalian and microbial cell culture ■

systems.FoamAway™ Irradiated is specifically designed to offer high

performance and convenience in combating foam in your cell culture system. It is prediluted, presterilized, and packaged to connect directly to your bioreactor with no further processing required.

FoamAway™ Irradiated consists of a 30% simethicone emulsion diluted with WFI (water for injection)-grade purified water to a final concentra-tion of 3%. This material is then packaged into flexible media bags and irradiated. Gamma-irradiation sterilization of the FoamAway™ product family has been validated to a sterility assurance level (SAL) of 10-6. The radiation validation study was based on the Association for the Advance-ment of Medical Instrumentation (AAMI) Technical Information Report (TIR) No. 33:2005 standard, entitled “Sterilization of Health Care Products—Radiation—Substantiation of a Selected Sterilization Dose—Method VDmax”. This process validates a sterilization dose based on the determination of the product bioburden and comparison of that bioburden information to a model population having a defined resistance to radiation. AAMI TIR No. 33:2005 was designed to meet the requirements of ANSI/AAMI/ISO 11137, entitled “Sterilization of Health Care Products—Radiation Sterilization”. Please note that FoamAway™ Irradiated cannot be filtered due to the size of the simethicone particles.Recommended storage conditions: room temperature; shake well before use; do not filter.


500 ml in 1 L bag 006-0096BC

2.5 L in 5 L bag 006-0096BA

For more information about additional components exclusively available through Invitrogen, such as an alternative for recombinant human insulin, please contact our Custom Products Services group or www.invitrogen.com/limitededition.


FREE FoamAway™ Irradiated AOFReady-to-use, animal origin–free reagent for both antifoaming and ■

defoaming applications Can be used with both mammalian and microbial cell culture ■

systems Formulation and media bag contact layer contain no components ■

derived from human or animal sourcesFoamAway™ Irradiated AOF is specifically designed to offer high performance and convenience in combating foam in your cell culture system. It is prediluted, presterilized, and packaged to connect directly to your bioreactor with no further processing required.

FoamAway™ Irradiated AOF consists of an animal origin–free 30% simethicone emulsion diluted with WFI (water for injection)-grade purified water to a concentration of 3%. This material is then packaged into flexible media bags and irradiated. Gamma-irradiation sterilization of the FoamAway™ product family has been validated to a sterility assurance level (SAL) of 10–6. The radiation validation study was based on the Association for the Advancement of Medical Instrumentation (AAMI) Technical Information Report ( TIR) No. 33:2005 standard, entitled “Sterilization of Health Care Products—Radiation—Substantiation of a Selected Sterilization Dose—Method VDmax”. This process validates a sterilization dose based on the determination of the product bioburden and comparison of that bioburden information to a model population having a defined resistance to radiation. AAMI TIR No. 33:2005 was designed to meet the requirements of ANSI/AAMI/ISO 11137, entitled “Sterilization of Health Care Products—Radiation Sterilization”. Please note that FoamAway™ Irradiated AOF cannot be filtered, due to the size of the simethicone particles.Recommended storage conditions: room temperature; shake well before use; do not filter.For more information, visit www.invitrogen.com/foamaway.


500 ml in 1 L bag A1036902

2.5 L in 5 L bag A1036901





Biological BuffersThe growth of mammalian cells in culture media depends on both the pH of the media and the species of buffer present. Also, a high buffer capacity with a low buffer concentration is needed to avoid an undesirable fluctuation of pH due to tissue metabolism. Fluctuation of pH may lead to inhibition of growth or have other undesirable effects on cell parameters.

Disadvantages and limitations of bicarbonate/CO2 and phosphate buffers are well known, and Tris buffers are cytotoxic in certain cases. HEPES is the most generally used zwitterionic buffer.

Improved pH control between pH 6.7 and 8.4 may be obtained when 10 to 25 mM HEPES is incorporated into culture media.However, as buffer species vary in their toxicity to different types of cells, care must be taken in choosing buffers and their concentrations for use

with new cell lines.

HEPES Buffer Solution (1 M)pH: 7.2 to 7.5.pKa: 7.3 at 37°C.Recommended storage conditions: 2°C to 8°C.Intended use: In vitro diagnostic (IVD).


20 ml 15630-049

100 ml 15630-056

20 × 100 ml (case) 15630-122

Contains 238.3 g/L prepared in distilled water.

HEPES Ultrapure(N-2-hydroxyethylpiperazine-N´-2-ethane sulfonic acid)C8H18N2O4SFormula weight: 238.Purity: >99.5%.Useful pH range: 6.8 to 8.2.Recommended storage conditions: 15°C to 30°C


1 kg 11344-041

Sodium Bicarbonate Solution, 7.5%7.5 g/100 mlRecommended storage conditions: 15°C to 30°C.


100 ml 25080-060


158


Cell Dissociation ProductsCell Dissociation BuffersCell Dissociation Buffer, enzyme free, Hanks’-based Cell Dissociation Buffer, enzyme free, PBS-basedCell dissociation buffers are membrane-filtered, isotonic, and enzyme-free aqueous formulations of salts, chelating agents, and cell-conditioning agents in either Ca2+- and Mg2+-free Hanks’ balanced salt solution or Ca2+- and Mg2+-free phosphate-buffered saline. They are suitable for gentle dissociation of mammalian cells from support substrates and each other, and for studies requiring intact cell-surface proteins such as ligand binding, flow cytometry, and immunohistochemistry.1,2 These buffers are not recommended for routine passage of strongly adherent cell types.Performance and quality testing: Buffers are membrane filtered and mycoplasma tested. Cell integrity after dissociation with buffers is measured by a plating efficiency assay and a serotonin receptor function assay.Recommended storage conditions: 15°C to 30°C.

References1. Julius, D., Livelli, T.J., Jessell, T.M., and Axel, R. (1989) Science 244, 1057.2. Livelli, T.J. (1989) FOCUS® 11, 74.


100 ml (Hanks’-based) 13150-016

100 ml (PBS-based) 13151-014

Collagenase, lyophilized(Clostridiopeptidase A). Prepared from Clostridium histolyticum.E.C. number: 3.4.2.4.3Activity: ≥125 units/mg weight. One unit will liberate 1 µmol amino acid from collagen, expressed as L-leucine, in 18 hr at 37°C.Recommended storage conditions: 2°C to 8°C.


500 mg 17018-029

Collagenase Type IPrepared from Clostridium histolyticum for dissociation of tissues. Suggested for epithelial, lung, fat, and adrenal tissue cell preparations.Recommended storage conditions: 2°C to 8°C.


1 g 17100-017

Collagenase Type IIPrepared from Clostridium histolyticum for dissociation of tissues. Suggested for preparation of cells from liver, bone, thyroid, heart, and salivary gland tissue.Recommended storage conditions: 2°C to 8°C.


1 g 17101-015

Collagenase Type IVPrepared from Clostridium histolyticum for dissociation of tissues from pancreas. For isolation of pancreatic islets.Recommended storage conditions: 2°C to 8°C.


1 g 17104-019

DispasePrepared from Bacillus polymyxa. For gentle dissociation of animal tissues to release individual cells.Recommended storage conditions: 2°C to 8°C.


5 g 17105-041





Trypsin SolutionsGibco® trypsin solutions are used widely for dissociation of tissues and cell monolayers. We can provide cell culture dissociating solutions that will perform consistently, batch after batch.

The biochemical assays performed on Trypsin (1:250) determine both the trypsin-specific activity and the level of certain copurified enzymes that influence cell removal and viability. All trypsin solutions are prepared from porcine parvovirus–tested and mycoplasma-tested materials.

Gibco® trypsin solutions are prepared under controlled conditions, using accelerated manufacturing procedures to minimize loss of enzyme activity from denaturation and autodegradation.Raw material quality control. Trypsin powdered raw material is subjected to standard assays for solubility, loss of weight on drying, residue on ignition, and trypsin activity as outlined in the current edition of the U.S. Pharmacopeia, National Formulary (USP/NF).

Accepted valuesActivity: One USP trypsin unit is the activity causing a change in absorbance of 0.003/min in the presence of N-benzoyl-L-arginine ethyl ester hydrochloride at 25°C, pH 7.6. Trypsin (1:250) has an activity such that 1 g will digest (hydrolyze) 250 g of casein under the conditions of the USP test for protease activity of pancreatin.Porcine parvovirus: None detected.Mycoplasma: None detected.Amylase activity: Acceptable.Nucleotidase activity: Acceptable.Final product quality control: GIBCO® enzyme preparations are tested to confirm the absence of bacterial and fungal contamination, employing protocols referenced in the current USP. The pH and osmolality of all batches are monitored using calibrated measuring equipment to verify conformance with product specifications.

In vitro bioassays are performed on the final product to verify the ability of the trypsin solutions to remove an adherent cell line from the substratum while preserving cellular integrity. The cell line selected for this assay is a human transformed cell line. The trypsin-dependent cell-removal times must be within a range established for each cell line. Following trypsinization, cells are replated and morphology is monitored after 18 to 24 hr. Trypsin removal and replating are tested in parallel with a reference trypsin solution. For more information about GIBCO® enzyme preparations, contact Invitrogen Technical Support.

Powdered TrypsinWe offer trypsin preparations as nonsterile powders.

Gamma-Irradiated Liquid and Powdered TrypsinUpon request, trypsin can be gamma-irradiated. We have validated the use of gamma-irradiation to inactivate certain porcine viruses which may be present in trypsin. Our studies indicate that an irradiation dose of 25 kGy will inactivate virus while preserving the biological performance in cell culture and the trypsin enzymatic activity. Additionally, osmolality and pH remain unchanged. For further information about our validation results, contact the Invitrogen Technical Support.

Trypsin Replacement EnzymesTrypLE™ trypsin replacement enzyme is a microbially produced alternative to animal trypsin. It is manufactured in a controlled fermentation process that is completely free of animal- and human-derived components. The process produces an exceptionally pure reagent that is gentle on cells. Inactivation with trypsin inhibitors is not required. GIBCO® TrypLE™ enzyme can be stored refrigerated and is stable at room temperature for 6 months. Visit www.invitrogen.com/trypleexpress for more information.


FREE TrypLE™ Select Stable Trypsin Replacement Enzyme (1X), liquid

An animal origin–free cell dissociation enzyme. This recombinant enzyme is an alternative to animal trypsin for the dissociation of adherent mammalian cells from plasticware. TrypLE™ Select is manufactured for the industrial market and has release testing for both endotoxin and enzyme activity. This material also has a Drug Master File on record.

Formulated in D-PBS with 1 mM EDTA. ■

Stable at 15°C to 30°C for 6 months. ■

Recommended storage conditions: 2° to 8°C.


100 ml 12563-011

500 ml 12563-029

Contains no phenol red.


160



FREE TrypLE™ Express, Stable Trypsin Replacement Enzyme (1X), liquid

A pure enzyme for gentler cell dissociation. This recombinant enzyme is an alternative to animal trypsin for the dissociation of adherent mammalian cells from plasticware. TrypLE™ Express is animal origin–free and thus ideal for both serum and serum-free cultures.

Formulated in D-PBS with 1 mM EDTA. ■

Stable at 15°C to 30°C for 6 months. ■

Recommended storage conditions: 2°C to 8°C.


100 ml 12605-010

500 ml 12605-028

20 × 100 ml (case) 12605-036

5 L 12605-093

Contains phenol red.

Quantity Cat. no.

100 ml 12604-013

500 ml 12604-021

5 L 12604-054

20 × 100 ml (case) 12604-039


Trypsin, 0.25% (1X), liquidPorcine parvovirus and mycoplasma tested. Source: Porcine.Recommended storage conditions: –5°C to –20°C.


100 ml 25050-014

500 ml 25050-030

Contains phenol red, 2.5 g/L of trypsin (1:250) in Hanks’ Balanced Salt Solution without CaCl2, MgCl2•6H2O, or MgSO4•7H2O.

Trypsin, 2.5% (10X), liquidPorcine parvovirus and mycoplasma tested. Source: Porcine.Dilution: Aseptically prepare 1X solution in balanced salt solution without Ca++ or Mg++.Final concentration: 2.5 g/L of trypsin (1:250).Recommended storage conditions: –5°C to –20°C.


100 ml 15090-046

Contains 25 g/L of trypsin (1:250) and 8.5 g/L of NaCl but no phenol red.

Trypsin-EDTA (0.05% Trypsin, EDTA • 4Na) (1X), liquidPorcine parvovirus and mycoplasma tested. Recommended storage conditions: –5°C to –20°C.


100 ml 25300-054

10 × 500 ml (case) 25300-104

20 × 100 ml (case) 25300-096

500 ml 25300-062

Contains phenol red, 0.5 g/L of trypsin (1:250) and 0.2 g/L of EDTA•4Na in Hanks’ Balanced Salt Solution without CaCl2, MgCl2•6H2O, or MgSO4•7H2O.

Trypsin-EDTA (0.5% Trypsin, EDTA•4Na) (10 X), liquidPorcine parvovirus and mycoplasma tested. Dilution: Aseptically prepare 1X solution in balanced salt solution without Ca++ or Mg++.Final concentration: 0.5 g/L of trypsin and 0.2 g/L of EDTA•4Na (1:250).Recommended storage conditions: –5°C to –20°C.


100 ml 15400-054

Contains 5.0 g/L of trypsin (1:250), 2.0 g/L of EDTA•4Na, and 8.5 g/L of NaCl but no phenol red.

Trypsin-EDTA (0.25% Trypsin, EDTA•4Na) (1X), liquidPorcine parvovirus and mycoplasma tested. Recommended storage conditions: –5°C to –20°C.


100 ml 25200-056

20 × 100 ml (case) 25200-114

500 ml 25200-072

Contains phenol red, 2.5 g/L of trypsin (1:250), and 0.38 g/L of EDTA•4Na in Hanks’ Balanced Salt Solution without CaCl2, MgCl2•6H2O, or MgSO4•7H2O.





Trypsin (1:250), powderPorcine parvovirus and mycoplasma tested.Activity: 1 g of trypsin will digest 250 g of casein substrate under the conditions of the USP monographs for protease activity of pancreatin (25°C for 10 min at pH 7.6).1

Recommended storage conditions: 2°C to 8°C.

Reference1. United States Pharmacopeia.


100 g 27250-018

Trypsin Inhibitor, soybeanActivity: >7,000 BAEE units of inhibition per mg.Recommended use: 0.25 to 0.5 mg/ml in PBS to inactivate an equal volume of trypsin.Recommended storage conditions: –5°C to –20°C.


1 g 17075-029

Versene 1: 5,000 (1X), liquidConcentration: 1X = 0.2 g/L = 0.48 mM.Recommended storage conditions: 2°C to 8°C.


100 ml 15040-033

Contains 0.2 g/L EDTA•4Na in phosphate-buffered saline.

Distilled Water

Water, distilledMembrane filtered and endotoxin screened.Recommended storage conditions: 15°C to 30°C.


100 ml 15230-071

20 × 100 ml (case) 15230-196

500 ml 15230-089

10 × 500 ml (case) 15230-204

1 L 15230-097

6 × 1000 ml 15230-001

5 L 15230-253

10 L 15230-261

new WFI for Cell Culture (Water for Injection)

Pure Alwaysgibco® WFI for Cell Culture is high quality, cell culture grade water, research use only. Every batch of gibco® WFI for Cell Culture is membrane-filtered to 0.1 µm and undergoes strict quality-control testing for sterility, endotoxin levels, conductivity, osmolality, oxidizable substances, and pH. gibco® WFI for Cell Culture meets the USP monograph for water for injectionRecommended storage conditions: gibco® WFI for Cell Culture, USP water for injection, research use only has a 12 month shelf life at 15-30°C.


500ml A1287-301

1000ml A1287-302

10X500ml A1287-303

6x1L A1287-304


162


Attachment Factors

Fibronectin, Human PlasmaFibronectin is a large cell-surface and plasma protein that exhibits structural and adhesive properties in cell-associated fibrillar matrices. Fibro nectin is one of the primary cell adhesion molecules. Human plasma fibronectin is isolated from human plasma by affinity chromatography. Fibroncetin has a molecular weight of 440,000 and is made up of two 220,000 DA polypeptides. Human fibronectin is filtered (0.2 µm) and supplied lyophilized.Performance and quality testing: Purity is determined by SDS-PAGE. Human fibronectin is assayed for its ability to support cell attachment and spreading, and screened to confirm absence of myco plasmas or bacterial contamination.Recommended storage conditions: –5°C to –20°C.Notes: Do not store in dilute solution. Avoid repeated freezing and thawing.Caution: Human-origin materials are nonreactive (donor level) for anti-HIV 1 and 2, anti-HCV, and HBs Ag. Handle in accordance with established biosafety practices.


5 mg 33016-015

Natural Mouse LamininNatural Mouse Laminin is purified from the Engelbreth-Holm-Swarm (EHS) sarcoma and is provided in Tris-buffered saline.Performance and quality testing: Purity is determined by SDS-PAGE. Mycoplasma content was determined to be negative. Functional performance is verified by cell attachment.Recommended storage conditions: –20°C.Notes: Do not store in dilute solution. Avoid repeated freezing and thawing.


1 mg 23017-015





Growth Supplements250X Cholesterol Lipid Concentrate250X Cholesterol Lipid Concentrate is a chemically defined, protein-free supplement used for growing NS0 and other lipid dependent cell lines to high densities while supporting growth and protein expression without complicating downstream purification. The supplement can be added to 1X media such as CD Hybridoma Medium and filtered with minimal loss of lipids. Custom formulations and packaging configurations as well as supplements used in feeding strategies are available through our Custom Products Services group.Recommended storage conditions: 2°C to 8°C, in the dark.

Reference1. Gorfien, S., Paul, W., Walowitz, J., Keem, R., Biddle, W., and Jayme, D., Growth of

NS0 cells in protein-free, chemically defined medium (2000) Biotechnol. Prog. 16: 682-687.


100 ml 12531-018

Related products PageHybridoma products . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 106

Chemically Defined Lipid ConcentrateChemically Defined Lipid Concentrate is a concentrated lipid emulsion designed to reduce or replace fetal bovine serum in cell culture media for a wide variety of applications, including growth and maintenance of CHO, hybridoma, and insect cells in culture; monoclonal antibody production by hybridomas; and viral expression in insect cells. The components of this medium supplement are chemically defined.For product formulation please refer to the tables at the end of this chapter.

Insect cell culture tested.Recommended storage conditions: 2°C to 8°C, in the dark.Attention: This material is hazardous. All persons using this product should review the Material Safety Data Sheet before handling.


100 ml 11905-031

AlbuMAX® ILipid-rich bovine serum albumin for cell culture.1 It is chromatographically purified and has an IgG content ≤ 0.1%.Recommended storage conditions: 2°C to 8°C.


25 g 11020-021

1 kg 11020-039

AlbuMAX® IILipid-rich bovine serum albumin for cell culture.1 It is chromatographically purified and has an IgG content <1.0%.Recommended storage conditions: 2°C to 8°C.

Reference1. Airey, P., Benny, G., Swartzwelder, F., and Conrad, D. (1991) FOCUS® 13, 2.


25 g 11021-029

100 g 11021-037

1 kg 11021-045

Bovine Albumin Fraction V Solution (7.5%)7.5 g/100 mlPrepared with Bovine Serum Albumin Fraction V in phosphate-buffered saline.Recommended storage conditions: 2°C to 8°C.


100 ml 15260-037


164


B-27 Serum-free SupplementsB-27 Serum-free Supplements are 50X liquid, serum-free supplements for growth and long-term viability of hippocampal neurons.1,2 These supplements are used in combination with a basal medium such as Neuro basal™ Medium.2

B-27 Supplement Minus AO is the complete B-27 Supplement without 5 antioxidants (vitamin E, vitamin E acetate, superoxide dismutase, catalase, and glutathione) that would interfere with studying free-radical damage to neurons.

B-27 Supplement Minus Vitamin A is the complete B-27 Supplement without vitamin A. The omission of vitamin A in this supplement prevents neural differentiation of stem cells.Recommended storage conditions: –5°C to –20°C.

References1. Brewer, G.J., Torricelli, J.R., Evege, E.K., and Price, P.J. (1993) J. Neuroscience Res.

35, 567.2. Brewer, G.J., Torricelli, J.R., Evege, E.K., and Price, P.J. (1994) FOCUS® 16, 6.


B-27 Serum-free Supplement (50X), liquid 10 ml 17504-044

B-27 Supplement Minus AO (50X), liquid 10 ml 10889-038

B-27 Supplement Minus Vitamin A (50X), liquid 10 ml 12587-010

Related products PageNeurobiology products . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .114

G-5 Supplement (100X), liquidG-5 Supplement is a serum-free supplement for the growth and expression of glial cells (normal and tumor) of astrocytic phenotype.For product formulation please refer to the tables at the end of this chapter.Recommended storage conditions: –5°C to –20°C.


1 ml 17503-012


Insulin, Human Recombinant, Zinc4 mg/mlPotency: (dry basis) ≥27 USP units/mg.Suggested range for supplementation: 2 to 20 µg/ml.Recommended storage conditions: –5°C to –20°C.


5 ml 12585-014

Insulin-Transferrin-Selenium Supplements (ITS)Gibco® Insulin-Transferrin-Selenium supplements reduce the fetal bovine serum (FBS) concentration required for routine maintenance and low-density attachment of many cell types.

Two supplements are available for use with noncomplex media such as RPMI 1640 or Earle’s Minimum Essential Medium: Insulin-Trans-ferrin-Selenium-G for general use and Insulin-Transferrin-Selenium-A for adherent cell cultures. In addition, a third supplement, Insulin-Transferrin- Selenium-X, is available for use with complex media that contain sodium pyru vate, such as F-12 Nutrient Mixture, Dulbecco’s Minimum Essential Medium/F-12 Nutrient Mixture, or Earle’s Minimum Essential Medium. All of these supplements are supplied at 100X and should be added to media at a ratio of 10 ml per liter of medium. Initially, FBS must be added at a concentration of 2% to 4% to achieve growth comparable to the same medium supplemented with 10% FBS; FBS concentration can be reduced further following initial adaptation.Performance testing: Not tested to determine plating efficiency.

Insulin-Transferrin-Selenium-G Supplement (100X)Prepared in Earle’s Balanced Salt Solution (EBSS).For use in general culture applications.Recommended storage conditions: 2°C to 8°C.


10 ml 41400-045


Insulin-Transferrin-Selenium-A Supplement (100X)Prepared in EBSS.For use in adherent (anchorage-dependent) cultures.Recommended storage conditions: 2°C to 8°C.


10 ml 51300-044






Insulin-Transferrin-Selenium Supplement Formulations

SupplementInsulin

(1.0 g/L)

Sodium selenite

(0.67 mg/L)Transferrin (0.55 g/L)

Sodium pyruvate (11.0 g/L)

Ethanolamine (0.2 g/L)

ITS-G ● ● ●

ITS-A ● ● ● ●

ITS-X ● ● ● ●

Notes: Supplements have been prepared in EBSS without phenol red. All formulations are 100X.

Insulin-Transferrin-Selenium-X Supplement (100X)Prepared in EBSS.For use with complex media that contain sodium pyruvate.Recommended storage conditions: 2°C to 8°C.


10 ml 51500-056

Contains no phenol red or sodium pyruvate.

Transferrin, Bovine (APO form), lyophilizedIron <50 µg/g protein.Suggested range for supplementation: 0.5 to 50 µg/ml.Recommended storage conditions: 2°C to 8°C.


1 g 11108-016

Transferrin, Bovine (holo form), lyophilized90% iron saturated.Suggested range for supplementation: 0.5 to 50 µg/ml.Recommended storage conditions: 2°C to 8°C.


100 mg 11107-018

25 g 11107-047

Transferrin, Bovine (partially iron loaded), lyophilizedIron content 400 to 500 µg/g protein.Suggested range for supplementation: 0.5 to 50 µg/ml.Recommended storage conditions: 2°C to 8°C.


1 g 11105-012

Lactalbumin Hydrolysate, powder (extra soluble)Tissue-culture grade.Recommended storage conditions: 15°C to 30°C.


5 kg 11800-042

N-2 Supplement (100X), liquidN-2 Supplement is a serum-free supplement for the growth and expression of post-mitotic neurons and tumor cells of neuronal phenotype.For product formulation please refer to the tables at the end of this chapter.Recommended storage conditions: –5°C to –20°C.


5 ml 17502-048



166


Hybridoma ReagentsWe offer high-quality reagents required for selecting hybridomas. Selection and confirmation of hypoxanthine guanine phosphoribosyl transferase (HGPRT )–deficient cells is achieved by use of the toxic base analog 8-azaguanine. HGPRT mutants will continue to pro liferate, whereas cells that possess HGPRT will die. HAT medium (medium supple-mented with hypoxanthine, aminopterin, and thymidine), which is used post-fusion as a selective medium, serves a dual purpose. Aminopterin, as an inhibitor of de novo nucleoside biosynthesis, is used to select against unfused myeloma cells and myeloma-myeloma fused partners. Hypoxanthine and thymidine supply preformed purines and pyrimi-dines for DNA synthesis by hybridomas via salvage pathways, as a result of the contribution of HGPRT by spleen cells. HT medium (medium supplemented with hypoxanthine and thymidine) is used as a rescue medium that continues providing preformed pur ines and pyrimidines downstream to overcome the effects of residual intracellular aminopterin.

Related products PageHybridoma products . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .106

250X Cholesterol Lipid Concentrate250X Cholesterol Lipid Concentrate is a chemically defined, protein-free supplement used for growing NS0 and other lipid-dependent cell lines to high densities while supporting growth and protein expression with-out complicating downstream purification. The supplement can be added to 1X media such as CD Hybridoma and filtered with minimal loss of lipids. Custom formulations and packaging configurations as well as supplements used in feeding strategies are available through our Custom Products Services group.Recommended storage conditions: 2°C to 8°C, in the dark.

Reference1. Gorfien, S., Paul, W., Walowitz, J., Keem, R., Biddle, W. and Jayme, D., Growth of

NS0 cells in protein-free, chemically defined medium (2000) Biotechnol. Prog. 16: 682–687.


100 ml 12531-018

Related products PageHybridoma products . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .106




100 ml 11905-031

HAT Supplement (50X), liquidHAT Supplement (50X) is a liquid mixture of sodium hypoxanthine, aminopterin, and thymidine. After dilution, HAT is suitable for use as a post-fusion selec-tive medium to eliminate unfused or self-fused HGPRT-myeloma cells. Hypoxanthine and thymidine supply preformed purines and pyrimidines for DNA synthesis by hybridomas via the salvage pathway that utilizes HGPRT contributed by the fused spleen cell. Ami nopterin, a folic acid antagonist, inhibits the de novo nucleoside bio syn thesis pathway.Concentration at 50X: 5 mM sodium hypoxanthine, 20 µM aminopterin, 0.8 mM thymidine.Recommended storage conditions: –5°C to –20°C.


100 ml 21060-017

HT Supplement (100X), liquidHT Supplement (100X) liquid is a mixture of sodium hypoxanthine and thymidine. After dilution, HT Supplement is suitable for use as a rescue medium. The supplement provides preformed purines and pyrimidines to overcome the effects of residual intracellular aminopterin. Once the de novo biosynthesis pathway for nucleosides has been reestablished, HT supplementation can be discontinued.Concentration at 100X: 10 mM sodium hypoxanthine, 1.6 mM thymidine.Recommended storage conditions: 2°C to 8°C.


50 ml 11067-030





Insect Medium Supplements

4% Agarose GelRecommended storage conditions: 2°C to 8°C.


40 ml 18300-012




100 ml 11905-031

Lactalbumin Hydrolysate, powder (extra soluble)Tissue-culture grade.Recommended storage conditions: 15°C to 30°C.


5 kg 11800-042

Pluronic® F-68, 10% (100X)Prepared in distilled water.

Insect cell culture tested.Recommended storage conditions: 15°C to 30°C.

Pluronic® is a registered trademark of BASF Corporation.


100 ml 24040-032

Yeastolate Ultrafiltrate (50X), liquidYeastolate Ultra fil trate is formulated specifically for use with IPL-41 Insect Medium to support the growth of Spodoptera frugiperda (Sf9). Prepared with 200 g/L of yeastolate in distilled water. Yeastolate Ultra fil trate is ultrafiltered using a 10,000 Da cutoff.

Insect cell culture tested.Performance testing: Performance tested in a growth assay using Sf9 cells.Recommended storage conditions: 2°C to 8°C, in the dark.


100 ml 18200-048

Related products PageInsect Media . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 36

Mycoplasma Detection

Yeast Extraction SolutionPrepared with distilled water. Yeast extract solution is autolyzed at 150 g/L.Recommended storage conditions: 2°C to 8°C.


100 ml 18180-059


168


Other Cell Culture Reagents

2-Mercaptoethanol (1,000X), liquid55 mM in D-PBS.Formula weight: 78.Recommended storage conditions: 2°C to 8°C.


50 ml 21985-023

Phytohemagglutinin (M Form) (PHA), liquidPhytohemagglutinin is a liquid extract of the red kidney bean, Phaseolus vulgaris. This extract is capable of inducing blastogenesis in vitro in various mammalian mononuclear cells. PHA is a crude sodium chloride extract of beans that have been homogenized in saline solution. Performance and quality testing: Each lot is assayed in a performance test system.Recommended storage conditions: –5° to –20°C.Attention: This material is hazardous. All persons using this product should review the Material Safety Data Sheet before handling.


10 ml 10576-015

Sodium Pyruvate Solution 100 mM (100X), liquid11,004 mg/L.Recommended storage conditions: 2°C to 8°C.Intended use: In vitro diagnostic (IVD).


100 ml 11360-039

20 × 100 ml 11360-088

Trypan Blue StainMembrane filtered and prepared in 0.85% saline.Recommended storage conditions: 15°C to 30°C.Attention: This material is hazardous. All persons using this product should review the Material Safety Data Sheet before handling.


100 ml 15250-061

MEM Vitamin Solution (100X), liquidPrepared in 0.85% saline. The vitamins in this solution are 100X the concentration in a standard MEM Medium.For product formulation please refer to the tables at the end of this chapter.pH: 5.4 to 7.7.Recommended storage conditions: –5°C to –20°C.Intended use: In vitro diagnostic (IVD).


100 ml 11120-037

D-Glucose (dextrose)USP grade.AnhydrousC6H12O6

Formula weight: 180.Recommended storage conditions: 15°C to 30°C.


1 kg 15023-021





MEM Amino Acids SolutionBase cat. no. 11130


Amino acids

L-Arginine 6,320.00

L-Cystine 1200.00

L-Histidine•HCl•H2O 2100.00

L-Isoleucine 2620.00

L-Leucine 2620.00

L-Lysine•HCl 3625.00

L-Methionine 755.00


L-Threonine 2380.00

L-Tryptophan 510.00

L-Tyrosine 1800.00

L-Valine 2340.00

G-5 Supplement Base cat. no. 17503

Component µg/ml

Insulin 500.00

Human transferrin 5000.00

Selenite 0.52

Biotin 1.00

Hydrocortisone 0.36

FGF 0.50

EGF 1.00

Chemically Defined Lipid ConcentrateBase cat. no. 11905

Component mg/L

Arachidonic acid 2.00

Cholesterol 220.00

DL-α-Tocopherol acetate 70.00

Linoleic acid 10.00

Linolenic acid 10.00

Myristic acid 10.00

Oleic acid 10.00

Palmitoleic acid 10.00

Palmitic acid 10.00

Pluronic® F-68 100,000.00

Stearic acid 10.00

Tween® 80 2200.00

Tween® is a registered trademark of ICI Ameri-cas, Inc. Pluronic® is a registered trademark of BASF Corporation.

Cell Culture Reagent Formulations

MEM Vitamin SolutionBase cat. no. 11120


NaCl 8500.00



Folic acid 100.00

i-Inositol 200.00

Nicotinamide 100.00

Pyridoxal HCl 100.00

Riboflavin 10.00

Thiamine HCl 100.00

N-2 Supplement Base cat. no. 17502

Component µg/ml

Insulin 500.00

Human transferrin 10000.00

Progesterone 0.63

Putrescine 1611.00

Selenite 0.52

MEM Non-Essential Amino Acids SolutionBase cat. no. 11140


Amino acids

L-Alanine 890.00

L-Asparagine 1320.00



Glycine 750.00

L-Proline 1150.00

L-Serine 1050.00


170


new PrestoBlue™ Cell Viability ReagentSave time with an incubation step as short as 10 minutes ■

Obtain high quality results with large assay window and z’ values ■

Experience convenience with an add-and-read homogenous format ■

Monitor living cells and perform downstream functional assays with ■

no cell lysis

PrestoBlue™ reagent is a resazurin-based solution that functions as a cell viability indicator by using the reducing power of living cells to quantitatively measure the proliferation of cells. The PrestoBlue™ reagent contains a cell-permeant compound that is blue in color and virtually nonfluorescent. When added to cells, the PrestoBlue™ reagent is modified by the reducing environment of the viable cell, turns red in color and becomes highly fluorescent. This change can be detected using fluorescence or absorbance measurements.

Assay WorkflowA 96-well plate containing the cells and the compounds to be tested is prepared. The PrestoBlue™ reagent is added directly to cells and incubated at 37oC for 10 minutes. The plate is then transferred to a fluorescence reader to measure signal. Results are evaluated by plotting signal versus compound concentration (Figure 1). In addition to this protocol format, the signal can alternatively be detected by absorbance or the assay can be run at room temperature.

PrestoBluetm reagent

➝ ➝ ➝ ➝

1. Add reagent to cells 2. Incubate 3. Read fluorescence 4. Process data

Figure 1. Data obtained using PrestoBlue™ reagent shows excellent linearity (R2 value of 0.9948) for cells plated at densities between 100 and 100,000 cells/well. The signal for 100 wells was greater than that from zero cells +/- 3 standard deviations, and the Z’ values were above 0.5 for cell densities of 391 cells/well or greater (Figure 2).





Reliable, high-quality results in 10 minutes

Figure 2. Excellent data obtained in just 10 minutes. Jurkat cells were plated in cell culture medium in a 384-well plate in quadruplicate starting at 100,000 cells/well with a two-fold serial dilution. Cells were incubated with PrestoBlue™ reagent for 10 minutes prior to fluorescence measurements..

Figure 3. Extremely sensitive detection. Jurkat cells were plated in cell culture medium in a 384-well plate in quadruplicate starting at 100,000 cells/well with a two-fold serial dilution. Fluorescence measurements were taken following 10 minutes of incubation and following 16 hours of incubation with PrestoBlue™ reagent.

Sensitive detection of cell viabilityAt 10 minutes of incubation, PrestoBlue™ reagent can detect as few as 98 cells/well, and the signal remains linear over the a broad dilution range. Following a 16-hour incubation with PrestoBlue™ reagent, as few as 12 cells/well exhibited a signal that was greater than that from zero cells +/- 3 standard deviations. Z’-factor values were above 0.5 for cell densities of 24 cells/well or greater (Figure 3). Note that at low cell densities, the signal-to-background ratio increases after 16 hours’ incubation.


PrestoBlue™ 25ml A13261

PrestoBlue™ 100ml A13262


172


Notes:


Chapter 7 Eukaryotic Cell LinesInsect Cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 174

D.Mel-2 Cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 174

High Five™ Cells. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 174

Sf9 Cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 175

Sf21 Cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 175

Mammalian Cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 177

293 Cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 177

FreeStyle™ 293 Expression Medium . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 177

CHO-S Cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 178

DG44 Cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 178

Primary Human Keratinocytes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 179

Cell Line Development Kits . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 180

new OptiCHO™ Express Kit . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 180

new OptiCHO™ Antibody Express Kit . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 181

new Gibco® Hepatocytes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 182

Chapter 7 Eukaryotic Cell Lines

174


Insect CellsD.Mel-2 CellsD.Mel-2 Cells (or Schneider S2 insect cells) were isolated from late-stage Drosophila melanogaster (fruitfly) embryos and are commonly used for the transient or stable expression of recombinant proteins.1 Drosophila cell lines have been historically cultured in stationary systems utilizing T-flasks and serum-supplemented Schneider’s Drosophila basal medium.2 Drosophila insect cells are not anchorage dependent and can be read-ily transferred between monolayer and suspension culture with no significant effect on cell growth; however, we recommend that D.Mel-2 seed stocks be maintained in suspension culture, which allows for more flexibility and ease of use.

Prepared from low-passage cells with documented lineage (15 to 20 ■

passages serum-free)1.5 × 10 ■ 7 total cells providedCan be thawed and used directly in suspension culture or used as a ■

monolayer for transfection applicationsProtocols for recovery, culture, and cryopreservation are provided on ■

the product insertApplication: For recombinant protein expression. Performance testing: Each lot is tested for growth and viability post-recovery from cryopreservation. The Master Seed Bank has been tested for viruses and mycoplasmas, and sterility and identity confirmed by isozyme analysis and karyotyping.Recommended storage conditions: –125°C to –200°C (liquid nitrogen).Caution: Handle material under Biosafety Level 1 containment procedures as defined by the Centers for Disease Control. DMSO has been identified as hazardous. All persons using this product should review the Material Safety Data Sheet before handling.

References1. Schneider, I. (1972) J. Embryol. Exp. Morph. 27, 363. 2. Grace, T.D. (1962) Nature 195, 788.


1.5 ml 10831-014


High Five™ CellsAdapted to Express Five® SFM (Cat. no. 10486-025)

High Five™ Cells (BTI-TN-5B1-4) are a clonal isolate derived from the parental Trichopulsia ni cell line and are commonly used for the expres-sion of recombinant proteins using the Baculovirus Expression Vector System (BEVS). The cells are adapted to serum-free suspension culture in Express Five® SFM, which saves significant time and expense associated with the adaptation of cultures to serum-free and/or suspension cultures. Express Five® SFM is a protein-free insect cell culture medium optimized to support maximum cell growth and recombinant gene expression.

Provided as a frozen stock in Express Five® SFM containing 7.5% ■

DMSO3 × 10 ■ 6 cells/ml providedProtocols for recovery, culture, and cryopreservation are provided on ■

the product insert Applications: For baculovirus propagation and recombinant protein expression. Performance testing: Each lot is tested for growth and viability post-recovery from cryopreservation and is also tested for the absence of mycoplasmas.Recommended storage conditions: –125°C to –200°C (liquid nitrogen).Caution: Handle material under Biosafety Level 1 containment procedures as defined by the Centers for Disease Control. DMSO has been identified as hazardous. All persons using this product should review the Material Safety Data Sheet before handling.

Important Licensing Information: This product is covered by a Limited Use Label License No. 66. By use of this product you accept the terms and conditions of relevant Limited Use Label License. For research purposes only.

Limited Use Label License No. 66: High Five™ Cells: The High Five™ cell line is patented by the Boyce Thompson Institute for Plant Research, Ithaca, New York, and is covered under U.S. Patent no. 5,300,435. The High Five™ cell line is sold for research purposes only. Commercial use requires a license from Boyce Thompson. For more information, please contact: Joyce L. Frank, Tel: 607-254-1220; Fax: 607-254-1242; E-mail: [email protected].


3 × 106 cells/ml B855-02



Eukaryotic Cell Lines




FREE Sf9 CellsAdapted to Sf-900™ III SFM (base cat. no. 12658), a protein-free and animal origin–free (AOF) medium

Sf9 cells, a clonal isolate derived from the parental Spodoptera frugiperda (fall armyworm) cell line IPLB-Sf21-AE, are commonly used to isolate and propagate recombinant baculovirus stocks and to produce recombinant proteins.

Can be scaled up for culture in bioreactors—provided as a frozen ■

stock in Sf-900™ III SFM containing 7.5% DMSOEach lot is tested for growth and viability post-recovery from ■

cryopreservation. The Master Seed Bank has been tested for viruses and mycoplasmas, and sterility and identity have been confirmed by isozyme analysis and karyotyping.

Storage conditions: –125°C to –200°C (liquid nitrogen).


1.5 × 107 cells/vial) 12659-017


FREE Sf21 CellsAdapted to Sf-900™ III SFM (base cat. no. 12658), a protein-free and animal origin–free (AOF) medium

Sf21 cells, isolated from Spodoptera frugiperda (fall armyworm) ovarian cells, are commonly used to isolate and propagate recombinant baculovirus stocks and to produce recombinant proteins.

Can be scaled up for culture in bioreactors—provided as a frozen ■

stock in Sf-900™ III SFM containing 7.5% DMSOEach lot is tested for growth and viability post-recovery from ■

cryopreservation. The Master Seed Bank has been tested for viruses and mycoplasmas, and sterility and identity have been confirmed by isozyme analysis and karyotyping.

Storage conditions: –125°C to –200°C (liquid nitrogen).


1.5 × 107 cells/vial) 12682-019

Sf9 CellsAdapted to Sf-900™ II SFM (base cat. no. 10902)

Sf9 cells are a clonal isolate derived from Spodoptera frugiperda (fall armyworm) IPLB-Sf21-AE cells and are used for transient or stable expression of recombinant proteins. Sf9 cells are adapted to serum-free suspension culture in Sf-900™ II SFM, which saves significant time and expense associated with the adaptation of cultures to serum-free and/or suspension cultures.1,2 Insect cells have been historically cultured in stationary systems utilizing T-flasks and serum-supplemented basal medium.3 Insect cells are generally not anchorage dependent, and we recommend that the Sf9 seed stocks be maintained in suspension culture, allowing for more flexibility and ease of use.


total passages and 15 to 20 passages serum-free)Provided as a frozen stock in Sf-900™ II SFM containing 7.5% DMSO ■

1.5 × 10 ■ 7 total cells providedCan be thawed and used directly in suspension culture for rapid ■

expansion of cell stocks, propagation of baculovirus stocks, and production of recombinant proteins, or used as a monolayer for transfection or plaque assay applicationsProtocols for recovery, culture, and cryopreservation are provided on ■

the product insertApplications: For baculovirus propagation and recombinant protein expression. Performance testing: Each lot is tested for growth and viability post-recovery from cryopreservation. The Master Seed Bank has been tested for viruses and mycoplasmas, and sterility and identity have been confirmed by isozyme analysis and karyotyping.Recommended storage conditions: –125°C to –200°C (liquid nitrogen).Caution: Handle material under Biosafety Level 1 containment procedures as defined by the Centers for Disease Control. DMSO has been identified as hazardous. All persons using this product should review the Material Safety Data Sheet before handling.

References1. Smith, G.E., Ju, G., Ericson, B.L., Moshera, J., Lahm, H., Chizzonite, R., and Summers,

M.D. (1985) Proc. Natl. Acad. Sci. USA 82, 8404.2. Vaughn, J.L., Goodwin, R.H., Tompkins, G.L., and McCawley, P. (1977) In Vitro 13,

213.3. Grace, T.D. (1962) Nature 195, 788.


1.5 ml 11496-015



176


Sf21 CellsAdapted to Sf-900™ II SFM (base cat. no. 10902)

Sf21 cells (IPLB-Sf21-AE) were isolated from Spodoptera frugiperda (fall armyworm) ovarian insect cells and are used for transient or stable expression of recombinant proteins, which saves significant time and expense associated with the adaptation of cultures to serum-free and/or suspension cultures.1 Insect cells have been historically cultured in station-ary systems utilizing T-flasks and serum supplemented basal medium.2 Insect cells are generally not anchorage dependent and we recommend that the Sf21 seed stocks be maintained in suspension culture, allowing for more flexibility and ease of use.

Prepared from low-passage cells with documented lineage (100 to ■

110 total passages and 15 to 20 passages serum-free)Provided as a frozen stock in Sf-900™ II SFM containing 7.5% DMSO ■


expansion of cell stocks, propagation of baculovirus stocks, and production of recombinant proteins, or used as a monolayer for transfection or plaque assay applicationsProtocols for recovery, culture, and cryopreservation are provided on ■

the product insertApplications: For baculovirus propagation and recombinant protein expression. Performance testing: Each lot is tested for growth and viability post-recovery from cryopreservation. The Master Seed Bank has been tested for viruses and mycoplasmas, and sterility and identity have been confirmed by isozyme analysis and karyotyping.Recommended storage conditions: –125°C to –200°C (liquid nitrogen).Caution: Handle material under Biosafety Level 1 containment procedures as defined by the Centers for Disease Control. DMSO has been identified as hazardous. All persons using this product should review the Material Safety Data Sheet before handling.

References1. Vaughn, J.L., Goodwin, R.H., Tompkins, G.L., and McCawley, P. (1977) In Vitro 13,

213. 2. Grace, T.D. (1962) Nature 195, 788.


1.5 ml 11497-013






Mammalian Cells293 CellsAdapted to CD 293 Medium (Cat. no. 11913-019)

293-F and 293-H cells are clonal isolates derived from transformed embryonal human kidney cells.1,2 The 293-F strain is a fast-growing variant of the 293 cell line. The 293-H strain is a variant of the 293 cell line that demonstrates better adherence in monolayer culture and ease of use for plaque assays and other anchorage-dependent applications. The 293 parental cell line was selected for growth and transfection efficiency.2 Both 293-F and 293-H cell lines can be adapted to serum-free suspension culture in 293 SFM II or CD 293 Medium supplemented with L-glutamine for recombinant protein production and adenovirus propagation.

Prepared from low-passage serum-free adapted cells with ■

documented lineage (20 to 25 passages serum-free)Provided as a frozen stock in 293 SFM II containing 7.5% DMSO ■


expansion of cell stocksCan be cultured in monolayer for transfection with Lipofectamine™ ■

2000 Transfection Reagent 3

Protocols for recovery, culture, and cryopreservation are provided on ■

the product insertApplications: For recombinant protein expression, signal transduction, and receptor binding studies, and adenovirus and adeno-associated virus (AAV) propagation. Performance testing: Each lot is tested for cell growth and viability post-recovery from cryopreservation. The Master Seed Bank has been tested for viruses and mycoplasmas, and sterility and identity have been confirmed by isozyme analysis and karyotyping.Recommended storage conditions: –125°C to –200°C (liquid nitrogen).Caution: Handle material under Biosafety Level 2 containment procedures as defined by the Centers for Disease Control. DMSO has been identified as hazardous. All persons using this product should review the Material Safety Data Sheet before handling.

References1. Graham, F.L., Smiley, J., Russell, W.C., and Nairn, R. (1977) J. Gen Virol. 36, 59.2. Roy, L., Cates, S., Schifferli, K., Pichet, J.-P., Ciccarone, V., Bennett, S., and Hawley-

Nelson, P. (1999) FOCUS® 21, 62.3. Ciccarone, V., Chu, Y., Schifferli, K., Pichet, J.P., Hawley-Nelson, P., Evans, K., Roy,

L., and Bennett, S. (1999) FOCUS® 21, 54.


293-F Cells 1.5 ml 11625-019

293-H Cells 1.5 ml 11631-017

Related products Page293 expression products . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 93

FreeStyle™ 293 Expression MediumFreeStyle™ 293 Expression Medium is a chemically defined, protein-free medium specifically developed for the ability to support the growth and transfection of 293-F cells under suspension-type culture conditions. The medium is a complete, ready-to-use medium that has been supplemented with GlutaMAX™-I Supplement and is animal origin–free. FreeStyle™ 293 Expression Medium is able to save significant time and costs associated with adaptation of cell cultures to serum-free conditions.Recommended storage conditions: 2°C to 8°C, in the dark.

References1. Graham, F.L., Smiley, J., Russell, W.C., and Nairn, R. (1977) J. Gen. Virol. 36, 59.2. Harrison, T., Graham, F., and Williams, J. (1977) Virology 77, 319.3. Ciccarone, V., Chu, Y., Schifferli, K., Pichet, J.P., Hawley-Nelson, P., Evans, K., Roy, L.,

and Bennett, S. (1999) FOCUS® 21, 544. Epstein, D.A., Godwin, G.P., Gruber, D.F., and Grefrath, P.I. (1999) FOCUS® 21, 22.


6 × 1,000 ml (case) 12338-026


178


CHO-S CellsAdapted to CD CHO Medium (base cat. no. 10743)

CHO-S cells are a clonal isolate derived from Chinese hamster ovary (CHO K1) cells.1 The CHO-S parental cell line was selected for growth and transfection efficiency.3 The CHO-S cells are adapted to serum-free suspension culture in CD CHO Medium supplemented with L-glutamine and HT Supplement for transient or stable expression of recombinant proteins.2 CHO cells have historically been grown in serum supplemented medium. The use of serum-free media offers ease of downstream purification of expressed product, reduced risk of viral contamination and greater lot-to-lot consistency. CD CHO Medium is a chemically defined, protein-free medium formulated with no components of animal or human origin.


passages serum-free)Provided as a frozen stock in CD CHO Medium containing 7.5% ■

DMSO1.5 × 10 ■ 7 total cells providedCan be thawed and used directly in suspension culture for rapid ■

expansion of cell stocksCan be transfected in suspension culture using FreeStyle™ MAX ■

Transfection ReagentProtocols for recovery, culture, and cryopreservation are provided on ■

the product insertSee OptiCHO™ Express and OptiCHO™ Antibody Express Kits, pages ■ 170 and 171

Applications: For transfection, expression, and large-scale production of recombinant proteins.Performance testing: Each lot is tested for cell growth and viability post-recovery from cryopreservation. The Master Seed Bank has been tested for viruses and mycoplasmas, and sterility and identity have been confirmed by isozyme analysis and karyotyping.Recommended storage conditions: –125°C to –200°C (liquid nitrogen).Caution: Handle material under Biosafety Level 1 containment as defined by the Centers for Disease Control. DMSO has been identified as hazardous. All persons using this product should review the Material Safety Data Sheet before handling.

References1. D’Anna, J.A. (1996) Meth. Cell Sci. 18, 115.2. Schifferli, K., Jessee, J., and Ciccarone, V. (1999) FOCUS® 21, 16.


1.5 ml 11619-012

DG44 CellsPreadapted to CD DG44 Medium (Cat. no. 12610-010)*

DG44 cells, dihydrofolate reductase-deficient (DHFR–) a derivative of Chinese hamster ovary (CHO) cells, are routinely used to establish cell lines for production of recombinant protein. They were developed by Dr. Chasin at Columbia University using gamma rays to eliminate the entire DHFR locus. In nonmutant cells, DHFR is an essential enzyme for de novo synthesis of glycine, purines, and thymidylate. This allows DHFR– to be used as a dominant selectable marker and a gene amplifier for the expression of proteins in DHFR– cell lines. The DHFR– mutation in DG44 cells is stable and irreversible, which makes it a suitable mammalian cell line for production of recombinant proteins. GIBCO® offers a complete system for the growth and expansion of DG44 (DHFR–) cells in suspension, including the DG44 Cells and Media Kit (page 17).

Selected for superior cell growth and transfection efficiencies. Applications: For transfection, expression, and large-scale production of recombinant proteins.Performance testing: Each lot is tested for cell growth and viability post-recovery from cryopreservation. The Master Seed Bank has been tested for viruses and mycoplasmas, and sterility and identity have been confirmed by isozyme analysis and karyotyping.Recommended storage conditions: –125°C to –200°C (liquid nitrogen).Caution: Handle material under Biosafety Level 1 containment as defined by the Centers for Disease Control. DMSO has been identified as hazardous. All persons using this product should review the Material Safety Data Sheet before handling.

*CD DG44 Medium is a modification of CD CHO Media.





Primary Human KeratinocytesAdapted to Defined Keratinocyte-SFM (Cat. no. 10744-019)

Primary Human Keratinocytes are derived from normal human tissue and processed and frozen in Defined Keratinocyte-SFM. The cells are effectively adapted to the medium and will establish fully confluent cultures within five days following recovery from cryopreservation when our protocols are followed.

The cells are supplied as 1.1–1.6 × 10 ■ 6 total cells per vialThe lifespan of the cells is performance-tested for 16 population ■

doublings, with most lots demonstrating more than 20 doublingsProtocols for recovery and culture are provided on the product ■

insertApplication: For experimental protocols requiring monolayer cultures of undifferentiated human keratinocytes.Performance testing: Each lot is tested for cell growth and viability post-recovery from cryopreservation. All lots are screened for Hepatitis B and C, HIV, mycoplasmas, and microbial growth.Recommended storage conditions: –125°C to –200°C (liquid nitrogen).Caution: Handle material under Biosafety Level 1 containment as defined by the Centers for Disease Control. DMSO has been identified as hazardous. All persons using this product should review the Material Safety Data Sheet before handling.


1 ml 12332-011


180


Figure 1—Map of the pOptiVEC™-TOPO® cloning vector.

Ampicillin

pUC ori TK pA

DH

FR

PCMV IRES

TOPO

TOPO

CCCTTGGGA

AGGGTTCCC

pOptiVEC™-TOPO®

4.4 kb

Cell Line Development Kitsnew OptiCHO™ Express Kit

For expression vector construction, high-efficiency transfection of DG44 cells, and development of stable cell lines for protein expression.

The OptiCHO™ Express Kit is designed for efficient growth and transfection of dihydrofolate reductase–deficient (DHFR–) Chinese hamster ovary (CHO) DG44 cells in suspension culture. The kit provides a complete workflow protocol as well as the necessary components for efficient transfection of DHFR– DG44 cells in a serum-free environment and subsequent stable cell line development, including:

■ pOptiVEC™-TOPO® vector—a bicistronic cloning vector containing a DHFR– gene for stable cell line development in CHO-DG44 cells (Figure 1); the vector is TOPO® adapted and enables cloning of a PCR product containing your gene of interest with >85% efficiency


FREE ■ FreeStyle™ MAX Transfection Reagent—an animal origin–free transfection reagent offering high transfection efficiencies and consistent results

■ CD OptiCHO™ Medium—a chemically defined, protein-free medium (without hypoxanthine or thymidine) specifically designed for highdensity suspension cell culture of recombinant CHODG44 cells and secreted protein yield

■ CD DG44 Medium—a chemically defined, protein-free medium supplemented with hypoxanthine and thymidine to support optimal growth of DG44 cells in suspension culture

■ DG44 cells—preadapted to CD DG44 medium and selected for superior cell growth and transfection efficiencies

In addition, the OptiCHO™ Express Kit provides GIBCO® OptiPRO™ SFM for improved DNA-lipid complex formation, L-glutamine (provided separately for increased media stability), and Pluronic® F-68 to control shear forces in suspension cultures.Recommended storage conditions: Pluronic® F-68, 15°C to 30°C; CD DG44 Medium, OptiPRO™ SFM, CD OptiCHO™ Medium, and FreeStyle™ MAX Transfection Reagent, 2°C to 8°C, in the dark; L-glutamine, pOptiVEC™-TOPO® vector, CMV Forward Sequencing Primer, and EMCV IRES Reverse Sequencing Primer, –5°C to –20°C; One Shot® TOP10 Chemically Competent E. coli cells, –80°C; DG44 cells, liquid nitrogen.


Kit components, also available separately

pOptiVEC™-TOPO® TA Cloning Kit 1 kit 12744-017

CD DG44 Medium 1,000 ml 12610-010

FreeStyle™ MAX Transfection Reagent 1 ml 16447-100



Pluronic® F-68, 10% 100 ml 24040-032

L-glutamine-200 mM (100X) 100 ml 25030-081






Figure 1—Time savings throughout the cell line development process.

Potential for more than 6 months’ time saved using OptiCHO™ Express Kits

CHO cells

Expression vectors

Stable polyclonalpopulations

Protein productioncell lines

Adaptation

Clone selection

3 months

3 months

1–2 months

1–2 months

2–3 months

3 months

3 months

Current steps

Adaptation

Ampli�cation1 round

Total time: 16–19 months

OptiCHO™ cell engineering

CHO cells

Expression vectors

Stable polyclonalpopulations

Protein productioncell lines

Adaptation

Clone selection

0 months

0.5 months

1 month

1 month

2–3 months

3 months

0 months

Adaptation

Ampli�cation1 round

Total time: 7–9 months

new OptiCHO™ Antibody Express KitSerum-free cell line development and cloning for antibody expression.

The OptiCHO™ Antibody Express Kit is designed for faster serum-free cell line development and cloning for antibody expression in dihydrofolate reductase–deficient (DHFR–) Chinese hamster ovary (CHO) DG44 cells in suspension culture (Figure 1). The kit provides a complete workflow protocol and manual, as well as the necessary components for efficient transfection and cloning of DHFR– DG44 cells in a serum-free environment and subsequent development of a stable cell line for antibody production. The kit contains:

Two ■ expression vectors for light and heavy chains—the pOptiVEC™-TOPO® bicistronic cloning vector and pcDNA™3.3 TOPO® cloning vector (Figure 2); the vectors are TOPO® adapted to enable cloning of a PCR product containing your gene of interest with >85% efficiency

■ A N I M A LO R I G I N

FREE FreeStyle™ MAX Reagent—an animal origin–free transfection reagent offering high transfection efficiencies and consistent results

■ CD OptiCHO™ Medium—a chemically defined, protein-free medium (without hypoxanthine or thymidine) specifically designed for high density suspension culture of recombinant CHO DG44 cells and secreted protein yield

■ CD DG44 Medium—a chemically defined, protein-free medium supplemented with hypoxanthine and thymidine to support optimal growth of DG44 cells in suspension culture

■ DG44 cells—preadapted to CD DG44 medium and selected for superior cell growth and transfection efficiencies

In addition, the OptiCHO™ Antibody Express Kit provides GIBCO® OptiPRO™ SFM for improved DNA-lipid complex formation, Geneticin® Selective Antibiotic (G-418) for DHFR– selection, L-glutamine (provided separately for increased media stability), and Pluronic® F-68 to control shear forces in suspension cultures.Recommended storage conditions: Pluronic® F-68, 15°C to 30°C; CD DG44 Medium, OptiPRO™ SFM, and CD OptiCHO™ Medium, 2°C to 8°C, in the dark; FreeStyle™ MAX Transfection Reagent, 2°C to 8°C; Geneticin® Selective Antibiotic, –5°C to –20°C, or 2°C to 8°C; L-glutamine, pcDNA™3.3-TOPO® TA Cloning® Kit, and pOptiVEC™-TOPO® TA Cloning® Kit, –5°C to –20°C; One Shot® TOP10 Chemically Competent E. coli cells, –80°C; DG44 cells, liquid nitrogen.


Kit components sold separately

pcDNA™3.3-TOPO® TA Cloning® Kit 1 kit K8300-01

pOptiVEC™-TOPO® TA Cloning® Kit 1 kit 12744-017

CD DG44 Medium 1,000 ml 12610-010

FreeStyle™ MAX Transfection Reagent 1 ml 16447-100



Geneticin® Selective Antibiotic, liquid 100 ml 10131-027

Pluronic® F-68, 10% 100 ml 24040-032

L-Glutamine-200 mM (100X) 100 ml 25030-081


Figure 2—Maps of the pOptiVEC™-TOPO® and pcDNA™3.3-TOPO® cloning vectors.


182


new Gibco® HepatocytesPrimary hepatocytes isolated from the liver are effective tools for the in vitro evaluation of metabolism, drug-drug interactions, hepatotoxicity, and transporter activity. Gibco® hepatocytes have high viabilities and in vivo-like enzyme expression levels.

■ Extensive selection of lotsViabilities routinely >80% ■

Character ized for phase I and phase I I drug metabolizing ■

enzyme activitiesMultiple large lots available - ideal for reproducible long-term and ■

multi-site studies

Gibco® Cryopreserved Human and Animal HepatocytesAt Invitrogen, our hepatocyte isolationists are extensively trained in proper techniques to ensure optimal cell health. As a result, gibco® hepatocytes have high viabilities, in vivo-like enzyme expression levels, and if released as plateable cells, excellent confluencies that contribute to polarization and functioning cell-cell contacts

Extensive selection of lots ■

Viabilities routinely >80% ■

Characterized for phase I and phase II drug metabolizing, enzyme ■

activitiesMultiple large lots available—ideal for long-term studies across sites ■

Characterization and quality control: 10-point cell morphology check backed by photomicrographs—cell ■

membrane integrity, organelle size, presence of lipid droplets, nucleus size and shape, cytosolic clarity, cell shape, level of cell debris, cell excretion products, cell-cell contacts (plateable cells only)and reestablishment of bile canalicular networks (plateable cells only)Metabolic activity testing—ECOD, 7-HCG, 7-HCS; human hepatocytes ■

include—CYP1A2, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6, CYP2E1 CYP3A, FMOApplication qualification tests—attachment efficiency, monolayer ■

confluency fold induction with prototypical inducers, transporter uptake and efflux, in situ intrinsic clearanceAdditional characterization data—genotyping, optimal seeding ■

densities viability stabilities, donor demographics

Gibco® Human Cryopreserved Hepatocytes, Transporter Qualified

Contain functional membrane receptors and transporters ■

Facilitate effective transporter uptake and basolateral efflux (plated) ■

experimentsHave stringent release specs: ≥80% viability and ≥80% confluency ■

(plated cells)

Gibco® Human Hepatocytes, Induction QualifiedPrequalified for CYP1A2, CYP2B6 and CYP3A induction ≥80% viability ■

and ≥80% confluency (if plated cells)Minimum specific activities: ■

≥ 10-fold induction of CYP1A2≥ 5-fold induction of CYP2B6≥ 3-fold induction of CYP3A4

Gibco® Human Cryopreserved Hepatocytes, Plated Metabolism Qualified

Useful for the assessment of intrinsic clearance (CLint) in low turnover ■

compoundsPrequalified according to CLint of midazolam, tolbutamide, ■

dextromethorphan ≥80% viability and ≥75% attachment efficiency ■





Ordering InformationProduct Quantity Cat. No.

Human cryopreserved plateable hepatocytes (Each lot’s certificate of analysis has an exact cell count.)

Human Cryopreserved Plateable Hepatocytes, Transporter Qualified 4–8 million viable cells HMCPTS

Human Cryopreserved Plateable Hepatocytes, Transporter Qualified 9–12 million viable cells HMCPTL

Human Cryopreserved Plateable Hepatocytes, Induction Qualified 4–8 million viable cells HMCPIS

Human Cryopreserved Plateable Hepatocytes, Induction Qualified 9–12 million viable cells HMCPIL

Human Cryopreserved Plateable Hepatocytes, Metabolism Qualified 4–8 million viable cells HMCPMS

Human Cryopreserved Plateable Hepatocytes, Metabolism Qualified 9–12 million viable cells HMCPML

Human cryopreserved suspension hepatocytes

Human Cryopreserved Suspension Hepatocytes, Transporter Qualified 4–8 million viable cells HMCSTS

Human Cryopreserved Suspension Hepatocytes, Transporter Qualified 9–12 million viable cells HMCSTL

Human Cryopreserved Suspension Hepatocytes, Metabolism Qualified, Male 4–8 million viable cells HMCS1S

Human Cryopreserved Suspension Hepatocytes, Metabolism Qualified, Male 9–12 million viable cells HMCS1L

Human Cryopreserved Suspension Hepatocytes, Metabolism Qualified, Female 4–8 million viable cells HMCS2S

Human Cryopreserved Suspension Hepatocytes, Metabolism Qualified, Female 9–12 million viable cells HMCS2L

Human Cryopreserved Suspension Hepatocytes, Metabolism Qualified, Pooled Donors 4–8 million viable cells HMCS3S

Human Cryopreserved Suspension Hepatocytes, Metabolism Qualified, Pooled Donors 9–12 million viable cells HMCS3L

Animal suspension and plateable cryopreserved hepatocytes (Please inquire for minipig, guinea, and other custom species.)

Dog (Beagle) Cryopreserved Hepatocytes, Male 4–8 million viable cells DGCS10

Dog (Beagle) Cryopreserved Hepatocytes, Female 4–8 million viable cells DGCS20

Dog (Beagle) Cryopreserved Hepatocytes, Plateable Male 4–8 million viable cells DGCP10

Dog (Beagle) Cryopreserved Hepatocytes, Plateable Female 4–8 million viable cells DGCP20

Fish (Rainbow Trout) Cryopreserved Hepatocytes, Male 4–8 million viable cells TRCS10

Fish (Rainbow Trout) Cryopreserved Hepatocytes, Female 4–8 million viable cells TRCS20

Fish (Rainbow Trout) Cryopreserved Hepatocytes, Mixed Gender 4–8 million viable cells TRCS30

Monkey (Cynomolgus) Cryopreserved Hepatocytes, Male 4–8 million viable cells MKCS10

Monkey (Cynomolgus) Cryopreserved Hepatocytes, Female 4–8 million viable cells MKCS20

Monkey (Cynomolgus) Cryopreserved Hepatocytes, Plateable Male 4–8 million viable cells MKCP10

Monkey (Cynomolgus) Cryopreserved Hepatocytes, Plateable Female 4–8 million viable cells MKCP20

Mouse (CD-1) Cryopreserved Hepatocytes, Male 4–8 million viable cells MSCS10

Mouse (CD-1) Cryopreserved Hepatocytes, Female 4–8 million viable cells MSCS20

Mouse (CD-1) Cryopreserved Hepatocytes, Plateable Male 4–8 million viable cells MSCP10

Mouse (CD-1) Cryopreserved Hepatocytes, Plateable Female 4–8 million viable cells MSCP20

Rabbit (New Zealand White) Cryopreserved Hepatocytes, Male 4–8 million viable cells RBCS10

Rabbit (New Zealand White) Cryopreserved Hepatocytes, Female 4–8 million viable cells RBCS20

Rabbit (New Zealand White) Cryopreserved Hepatocytes, Plateable Male 4–8 million viable cells RBCP10

Rabbit (New Zealand White) Cryopreserved Hepatocytes, Plateable Female 4–8 million viable cells RBCP20

Rat (Sprague-Dawley) Cryopreserved Hepatocytes, Male 4–8 million viable cells RTCS10


184


Gibco® Fresh HepatocytesReliable supply from the industry’s largest sourcing network ■

Stringent release specs: ≥80% viability and ≥80% confluency (plated ■

cells)Ready for use—plated to your specifications, or provided in ■

suspensionPrimary human hepatocytes, under appropriate culture conditions, model metabolism, induction, and inhibition similar to the in vivo liver. Fresh human hepatocytes are used to test potential therapeutic compounds for their metabolic stability, metabolic profile, probability of hepatotoxicity, and ability to inhibit or induce metabolic enzymes and hepatic transporters. To achieve optimal in vitro results, primary hepatocytes must be carefully isolated to preserve their drug-metabolizing enzymes, transporters, and cellular activities. Our hepatocyte isolationists are experts in proper techniques to ensure optimal cell health. As a result, our fresh hepatocytes have high viabilities, excellent confluencies and in vivo-like enzyme expression levels.

Rapid Alert™ notificationsRapid Alert™ emails notify you right away about the impending availability of fresh primary human hepatocytes. When you receive a Rapid Alert™ email, just decide if you need hepatocytes, and contact us to place an order. Each Rapid Alert™ email contains: a summary of the donor’s demographics (gender, age, race, BMI, and history of alcohol, drugs, or medications) plate and suspension formats available for order a shipping date for the hepatocytes a phone number and email to place your order or request additional information.

To sign up for Rapid Alert™, access our online form at —www.invitrogen.com/hepatocytes


Chapter 8 Animal SeraFetal Bovine Sera (FBS) and other Sera for Cell Culture . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 186

General Animal Sera Information . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 186

The iMATCH™ MPA Sera Lot Matching Tool . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 186

FBS Selection Guide. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 187

Fetal Bovine Sera (FBS). . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 187

qualified . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 188

qualified, heat-inactivated . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 188

qualified, gamma irradiated . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 189

Specialty FBS Selection Guide. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 189

dialyzed. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 189

new charcoal stripped. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 189

ES cell qualified . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 189

new MSC qualified . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 190

ultra-low IgG FBS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 190

Other Sera Selection Guide . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 190

Newborn Calf Sera . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 191

Newborn Calf Serum . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 191

Newborn Calf Serum, heat-inactivated . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 191

Bovine Sera . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 191

Bovine Serum . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 191

Bovine Serum, heat-inactivated . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 191

Donor Bovine Serum . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 191

Donor Bovine Serum, with iron . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 191

Horse Sera . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 192

Horse Serum. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 192

Horse Serum, heat-inactivated . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 192

Other Sera . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 192

Chicken Serum . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 192

Goat Serum . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 192

Lamb Serum . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 192

Porcine Serum. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 192

Rabbit Serum . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 192

FBS Quality Control Test Comparison. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 193

Other Sera Quality Control Test Comparison . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 194

Final Product Quality Control. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 196

Performance Tests: Fetal Bovine Sera (FBS) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 197

Special Proccessing of Sera . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 200

Chapter 8 Animal Sera

186


Fetal Bovine Sera (FBS) and other Sera for Cell CultureA History of Value and Trust: A World leader TodayGlobal sourcing capabilities, multiple aseptic-processing facilities worldwide, batch size capabilities up to 2,000 litres, State-of-the-Art processing (including steam sterilisation in place/true pool technology), an unsurpassed commitment to both product quality and traceability, and a deep understanding of customers’ requirements, are just a few of the factors which have contributed over four decades to establishing Life Technologiestm as the major supplier of Fetal Bovine Serum (FBS) and other animal sera in the world today.

Vertical Integration of gibco® Sera Production Life Technologiestm oversees the preparation of gibco® sera through its entire process. Using specialised equipment, Standard Operating Procedures (which cover every step from blood collection to final product validation) and ongoing internal audits. Life Technologiestm ensures that quality is engineered into the product at every stage. This control over the entire process results in a consistent, high quality product with lower lot-to-lot variation.

Geographical Differentiation of FBS In response to the demand from the research community and biopharmaceutical companies. Life Technologiestm offers a choice of FBS differentiated by source. Our FBS product line feature products obtained from countries meeting USDA import requirements and/or E.U. import rules. All FBS products meet our rigorous quality control criteria. This differentiation allows the user to make an informed purchasing choice and offers price flexibility

Manufacture in Country of Origin In response to requests from the biopharmaceutical industry, Life Technologiestm now final-processes all normal sera of Australian, New Zealand and USA origins in the actual country of origin.

Traceability: your Proof of Serum Quality The Certificate of Analysis available for each lot of gibco® serum identifies the country of origin. This information is fully supported by extensive documentation that certifies each lot from collection through processing, testing, and delivery. This traceability is your proof of serum quality.

Batch Testing with ReservesMany of our customers purchase serum without pretesting with satisfactory results. However, we recognise the fact that some cell types are particularly sensitive to batch to batch variations in serum. Reserve testing allows customers to obtain a sample for testing in their own application with a reserve held on that batch. Serum is normally held on reserve for four weeks. If a longer test period is required, please advise us at time of reserve request.

The iMATCH™ MPA Sera Lot Matching tooliMATCH™ is a tool that our FBS specialists use to find you the most consistent or highest performing lot of serum available. iMATCH™ uses a proprietary algorithim – conducting multiparametric analysis of Certificate of analysis performance parameters to match your needs.The iMATCH™ ser vice is free of charge, only avai lable through Life Technologiestm and;

Saves time by reducing or eliminating serum testing ■

Makes serum choices based on performance, not source ■

Minimizes fluctuations in cell culture performance ■

Increases confidence in you search results ■

iMATCH™ is our promise to give you the best serum batch for your application… every time.

Shipping and Storage(a) All serum is routinely packed in dry ice for shipment. (b) We recommend that you store serum at -5°C to -20°C and use it before the expiration date indicated on the label.The plastic bottles may be stored at temperatures as low as -60°C to -70°C.(c) We also recommend that you avoid multiple freeze-thaws. If the serum is likely to be frozen and thawed frequently, dispense it aseptically into sterile containers in appropriate volumes and store it frozen in these containers. It is recommended that serum is thawed at +4°C to minimaise precipitation.

Handling of Bovine Origin Materials As with all materials of biological origin, Life Technologiestm recommends that all bovine products are treated as potentially harmful and that appropriate care is taken when handling and disposing of them.

Intended Use of ProductsThe products listed in this chapter are “for research use only”, except where otherwise denoted. “Research use only” products are not intended for diagnostic use. The safety and efficacy of these products in diagnostic or other clinical uses has not been established.

Products listed “for in vitro diagnostic use” are indicated by the symbol IVD and are medical devices subject to the requirements of Directive 98/79/EC of the European Parliament and of the Council of 27 October 1998 on in vitro diagnostic medical devices [Official Journal L 331 of 07.12.1998] and to the requirements of the Code of Federal Regulations Title 21, Part 820: Quality Systems Regulation. These products may also be used for research applications, and in appropriate circumstances, as raw material components in further manufacturing applications. It is the end user’s responsibility to qualify these products for their specific application. They have not been qualified nor are they intended for any animal or human therapeutic uses.

Label information will change to incorporate the “CE” mark on “for in vitro diagnostic use” products, and a glossary of other symbols found on the labels can be found on page 237.


Animal Sera



FBS Selection GuideProduct Description/usage guidelines Source Quantity Cat. no.

Certified FBS Undergoes special biochemical/hormonal profile and bacterial tests. Guaranteed low endotoxin (≤10 EU/ml) and low hemoglobin (≤15 mg/dl) levelsUse with your most sensitive cells

United States 100 ml 500 ml

16000-036 16000-044

Certified, heat-inactivated FBS

Heated for 30 min at 56°C with mixing to inactivate complementEspecially suited for immunological work


10082-139 10082-147

Qualified FBS Our most popular FBS product. High quality and exceptional value

Suitable for general applications, especially those that do not require defined biochemical/hormonal profiles or bacteriophage testing

Endotoxin ≤50 EU/ml—levels routinely less than 10 EU/ml

Hemoglobin ≤25 mg/dl


26140-087 26140-079

Australia 100 ml 500 ml

10099-133 10099-141

Countries that meet EU importation requirements (South America only)

100 ml

500 ml

10270-098

10270-106

Countries that meet EU importation requirements (excluding South America)

100 ml

500 ml

10106-151

10106-169

Qualified, heat-inactivated FBS

Heated for 30 min at 56°C with mixing to inactivate complement

Especially suited for immunological work


16140-063 16140-071

Australia 100 ml 500 ml

10100-139 10100-147


100 ml 500 ml

10500-056 10500-064


100 ml 500 ml

10108-157 10108-165

Qualified Gamma Irradiated FBS

Intended for application where virus contamination is of concern.Gamma Irradiated with a minimum dose of 25kGys. Intended for applications where virus contamination is of concern


100 ml 500 ml

10499-036 10499-044


100 ml 500 ml

10109-155 10109-165

Fetal Bovine Sera (FBS)

Fetal Bovine Serum, certifiedOrigin: United StatesEndotoxin level: ≤10 EU/ml. Hemoglobin level: ≤15 mg/dl.Recommended storage conditions: –5°C to –20°C.Intended use: In vitro diagnostic (IVD).


100 ml 16000-036

500 ml 16000-044

Fetal Bovine Serum, certified, heat-inactivatedOrigin: United StatesEndotoxin level: ≤10 EU/ml.Hemoglobin level: ≤15 mg/dl.Recommended storage conditions: –5°C to –20°C.Intended use: In vitro diagnostic (IVD).


100 ml 10082-139

500 ml 10082-147

Exceptions for FBS EU Approval origins.

Endotoxin <100 EU/ml and Hemoglobin<30 MG/dl (South America only)


188


Fetal Bovine Serum, qualifiedOrigin: United StatesEndotoxin level: ≤50 EU/ml (levels routinely ≤10 EU/ml).Hemoglobin level: ≤25 mg/dl.Recommended storage conditions: –5°C to –20°C.Intended use: In vitro diagnostic (IVD).


100 ml 26140-087

500 ml 26140-079

Fetal Bovine Serum, qualified, heat-inactivated Origin: United StatesEndotoxin level: ≤50 EU/ml (levels routinely ≤10 EU/ml).Hemoglobin level: ≤25 mg/dl.Recommended storage conditions: –5°C to –20°C.Intended use: In vitro diagnostic (IVD).


100 ml 16140-063

500 ml 16140-071

Fetal Bovine Serum, qualified Origin: AustraliaEndotoxin level: ≤50 EU/ml (levels routinely ≤10 EU/ml).Hemoglobin level: ≤25 mg/dl.Recommended storage conditions: –5°C to –20°C.Intended use: In vitro diagnostic (IVD).


100 ml 10099-133

500 ml 10099-141

Fetal Bovine Serum, qualified, heat-inactivated Origin: AustraliaEndotoxin level: ≤50 EU/ml (levels routinely ≤10 EU/ml).Hemoglobin level: ≤25 mg/dl.Recommended storage conditions: –5°C to –20°C.Intended use: In vitro diagnostic (IVD).


100 ml 10100-139

500 ml 10100-147

Fetal Bovine Serum, qualifiedOrigin: Countries meeting EU importation requirementsEndotoxin level: ≤100 EU/ml (levels routinely less than ≤20 EU/ml).Hemoglobin level: ≤30 mg/dl.Recommended storage conditions: –5°C to –20°C.Intended use: In vitro diagnostic (IVD).


100 ml 10270-098

500 ml 10270-106

100 ml 10106-151

500 ml 10106-169

Fetal Bovine Serum, qualified, heat-inactivatedOrigin: Countries meeting EU importation requirementsEndotoxin level: ≤100 EU/ml (levels routinely ≤20 EU/ml).Hemoglobin: ≤30 mg/dl.Recommended storage conditions: –5°C to –20°C.Intended use: In vitro diagnostic (IVD).


100 ml 10500-056

500 ml 10500-064

100 ml 10108-157

500 ml 10108-165


Animal Sera



Specialty FBS Selection GuideProduct Description/usage guidelines Source Quantity Cat. no.

Dialyzed FBS Dialyzed by tangential flow filtration utilizing 10,000 MW cutoff filtersPerformance tested for cloning and plating efficiencyIdeal for radiolabeling assays


26400-036 26400-044

Ultralow-IgG FBS IgG levels are less than 5 µg/ml, and the BVD antibody titer is low or not detectableSuitable for antibody production and veterinary applications


16250-086 16250-078

ES cell-qualified FBS Specially tested for the ability to sustain undifferentiated cellular morphology of embryonic stem cellsCrucial for the successful maintenance of embryonic stem cells


16141-061 16141-079

new MSC Qualified FBS Performance tested using a standard 14-day MSC CFU-f assayEach lot is tested against an in-house FBS reference standard using cells from a master cell bank of MSCs from normal bone marrow donors. This ensures lot-to-lot performance consistency.

Countries that meet USDA importation requirements (Mexico, Central America)

100 ml 500 ml

12662-011 12662-029

new Charcoal Stripped FBS

A serum product that will reduce lot-to-lot variability in hormone levels, which will help eliminate some of the influences steroids and other components have on cellsIdeal for customers doing cell culture work using immunoassay sys-tems and insulin assay methods

Countries that meet USDA importation requirements (Mexico, Central America)

50 ml 500 ml

12676-011 12676-029

Fetal Bovine Serum, dialyzedEndotoxin level: ≤50 EU/ml (levels routinely ≤10 EU/ml).Hemoglobin level: ≤25 mg/dl.Recommended storage conditions: –5°C to –20°C.Intended use: In vitro diagnostic (IVD).

Ordering InformationOrigin: United StatesQuantity Cat. no.

100 ml 26400-036

500 ml 26400-044

new Fetal Bovine Serum, charcoal strippedOrigin: Countries meeting USDA importation requirementsEndotoxin level: ≤50 EU/ml (levels routinely ≤10 EU/ml).Hemoglobin: ≤25 mg/dl.Recommended storage conditions: –5°C to –20°C.Intended use: For research use only.


50 ml 12676-011

500 ml 12676-029


100 ml 10109-155

500 ml 10109-163


100 ml 16141-061

500 ml 16141-079

Fetal Bovine Serum, qualified, gamma irradiated Origin: Countries meeting EU importation requirements stem cells.Endotoxin level: ≤100 EU/ml (levels routinely ≤20 EU/ml).Hemoglobin level: ≤30 mg/dl.Recommended storage conditions: –5°C to –20°C.Intended use: In vitro diagnostic (IVD).

Fetal Bovine Serum, ES cell qualifiedOrigin: United States. Specially tested for the ability to sustain undifferentiated cellular morphology of embryonic stem cells.Hemoglobin level: ≤25 mg/dl.Recommended storage conditions: –5°C to –20°C.Intended use: In vitro diagnostic (IVD).


190


Other Sera Selection GuideProduct Description/usage guidelines Source Quantity Cat. no.

Newborn Calf Serum Collected from calves typically 20 days old or younger New Zealand 100 ml 500 ml

16010-167 16010-159

Newborn Calf Serum, heat-inactivated

Collected from calves typically 20 days old or younger Heated for 30 min at 56°C with mixing to inactivate complement

New Zealand 100 ml 500 ml

26010-066 26010-074

Bovine Serum Collected from prime cattle with an age range of 12–36 months, but usually 24 months or younger


16170-086 16170-078

Bovine Serum, heat-inactivated

Collected from prime cattle with an age range of 12–36 months, but usually 24 months or younger. Heated for 30 min at 56°C with mixing to inactivate complement


26170-035 26170-043

Donor Bovine Serum Collected from donor cattle with an age range of 12–36 months, but usually 24 months or younger

New Zealand 500 ml 16030-074

Donor Bovine Serum, with iron

Collected from donor cattle with an age range of 12–36 months, but usually 24 months or younger. Ferric citrate complex added at 6.0 mg/dl

New Zealand 500 ml 10371-029

Horse Serum EIA tested (negative) from a donor herd New Zealand 100 ml 500 ml

16050-130 16050-122

Horse Serum, heat-inactivated

EIA tested (negative) from a donor herd Heated for 30 min at 56°C with mixing to inactivate complement


26050-070 26050-088

Chicken Serum Collected from healthy birds slaughtered for human consumption New Zealand 500 ml 16110-082

Goat Serum Collected from donor herd New Zealand 100 ml 500 ml

16210-064 16210-072

Lamb Serum Collected from animals younger than 1 year old New Zealand 500 ml 16070-096

Porcine Serum Collected from animals younger than 1 year old New Zealand 500 ml 26250-084

Rabbit Serum Collected from animals typically 6 months old or younger United States 100 ml 500 ml

16120-099 16120-107

new Fetal Bovine Serum, MSC qualified Origin: Countries meeting USDA importation requirementsSpecifically tested for the ability to attain enhanced mesenchymal stem cell clonal efficiency.Endotoxin level: ≤50 EU/ml (levels routinely ≤10 EU/ml).Hemoglobin level: ≤25 mg/dl.Recommended storage conditions: –5°C to –20°C.Intended use: In vitro diagnostic (IVD).


100 ml 12662-011

500 ml 12662-029

Related products PageStem cell research products . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .117

Fetal Bovine Serum, ultra-low lgG FBSOrigin: United StateslgG levels are less than 5 μg/ml, and the BVD antibody titer is low or not detectable.Endotoxin level: ≤50 EU/ml (levels routinely less than ≤10 EU/ml).Hemoglobin level: ≤25 mg/dl.Recommended storage conditions: –5°C to –20°C.Intended use: In vitro diagnostic (IVD).


100 ml 16250-086

500 ml 16250-078


Animal Sera



Bovine Sera (formerly Calf Sera)

Newborn Calf SeraNewborn Calf Serum, heat-inactivatedOrigin: New ZealandCollected from calves 20 days old or younger.Recommended storage conditions: –5°C to –20°C.Intended use: In vitro diagnostic (IVD).


100 ml 26010-066

500 ml 26010-074

Newborn Calf SerumOrigin: New ZealandCollected from calves 20 days old or younger.Recommended storage conditions: –5°C to –20°C.Intended use: In vitro diagnostic (IVD).


100 ml 16010-167

500 ml 16010-159

Bovine Serum Origin: New ZealandCollected from cattle 12–36 months of age.Recommended storage conditions: –5°C to –20°C.Intended use: In vitro diagnostic (IVD).


100 ml 16170-086

500 ml 16170-078

Donor Bovine Serum Origin: New ZealandCollected from cattle 12–36 months of age.Recommended storage conditions: –5°C to –20°C.Intended use: In vitro diagnostic (IVD).


500 ml 16030-074

Bovine Serum, heat-inactivatedOrigin: New ZealandCollected from cattle 12–36 months of age.Recommended storage conditions: –5°C to –20°C.Intended use: In vitro diagnostic (IVD).


100 ml 26170-035

500 ml 26170-043

Donor Bovine Serum, with iron Origin: New ZealandFerric citrate complex at 6.0 mg/dl.Collected from cattle 12–36 months of age.Recommended storage conditions: –5°C to –20°C.Intended use: In vitro diagnostic (IVD).


500 ml 10371-029


192


Horse SeraAll GIBCO® horse sera have been tested and found negative for equine infectious anemia (EIA).

Other Sera

Horse SerumOrigin: New ZealandEIA tested (negative) serum from a donor herd.Recommended storage conditions: –5°C to –20°C.Intended use: In vitro diagnostic (IVD).


100 ml 16050-130

500 ml 16050-122

Horse Serum, heat-inactivatedOrigin: New ZealandEIA tested (negative) serum from a donor herd.Recommended storage conditions: –5°C to –20°C.Intended use: In vitro diagnostic (IVD).


100 ml 26050-070

500 ml 26050-088

Chicken SerumOrigin: New ZealandRecommended storage conditions: –5°C to –20°C.Intended use: In vitro diagnostic (IVD).


500 ml 16110-082

Goat SerumOrigin: New ZealandRecommended storage conditions: –5°C to –20°C.Intended use: In vitro diagnostic (IVD).


100 ml 16210-096

500 ml 16210-072

Lamb SerumOrigin: New ZealandRecommended storage conditions: –5°C to –20°C.Intended use: In vitro diagnostic (IVD).


500 ml 16070-096

Porcine SerumOrigin: New ZealandRecommended storage conditions: –5°C to –20°C.Intended use: In vitro diagnostic (IVD).


500 ml 26250-084

Rabbit SerumOrigin: United States, New ZealandRecommended storage conditions: –5°C to –20°C.Intended use: In vitro diagnostic (IVD).


100 ml 16120-099

500 ml 16120-107


Animal Sera



FBS Quality Control Test Comparison

Test

Certified16000

Certified heat-

inactivated 10082

Qualified26140,10099,

Qualifiedheat-inactivated

16140, 10100Qualified

10270, 10106

Qualified heat-inactivated

10500, 10108Dialyzed

26400Ultralow-IgG

16250

Charcoal Stripped

12676ES Cell-Qualified

16141

MSC Qualified

12662

Bacteriophage tested ● ●

Biochemical profile ● ●

Bovine IgG (<5 µg/ml) ●

Determination of % oxy Hb (>70%) ● ●

Electrophoretic pattern ● ● ● ● ● ● ● ●

Endotoxin (EU/ml) ● ● ● ● ● ● ● ● ●

ES cell performance assays ●

MSC CFU-f assay ●

Glucose (<5 µg/ml) ●

Hemoglobin (mg/dl) ● ● ● ● ● ● ● ●

Hormone profile ● ● ●

Mycoplasmas ● ● ● ● ● ● ● ● ●

Osmolality ● ● ● ● ● ● ● ● ●

Performance tested ● ● ● ● ● ● ● ● ●

pH ● ● ● ● ● ● ● ● ●

Sf9 cell assay ● ●

Sterility testing (bacteria/fungi)

● ● ● ● ● ● ● ● ●

Tetracycline* ● ● ● ●

Total protein ● ● ● ● ● ● ● ● ●

Viral testing

Akabane virus ●

Bluetongue virus ● ● ● ● ● ● ●

Bovine adenovirus FA ● ● ● ● ● ● ●

Bovine parvovirus FA ● ● ● ● ● ● ●

Bovine respiratory syncytial virus FA

● ● ● ● ● ● ●

Bovine viral diarrhea virus FA

● ● ● ● ● ● ● ● ●

Bovine viral diarrhea neutralization assay ●

Cytopathic agents (e.g., IBR)

● ● ● ● ● ● ● ● ●

Hemadsorbing agents (e.g., PI-3)

● ● ● ● ● ● ● ● ●

Reovirus FA ● ● ● ● ● ● ●

Rabies virus FA ● ● ● ● ● ● ●


194


Other Sera Quality Control Test Comparison Base cat. no. 16010 26010 16170 26170 16030 10371 16050 26050 16110 16210 16070 26250 16120

Newborn Calf SerumNewborn Calf Serum,

heat-inactivated Bovine SerumBovine Serum

heat-inactivated Donor Bovine SerumDonor Bovine Serum

with iron Horse SerumHorse Serum,

heat-inactivated Chicken Serum Goat Serum Lamb Serum Porcine Serum Rabbit Serum

Cytotoxicity assay ● ● ●

Electrophoretic pattern ● ● ● ● ● ●√ ● ● ● ● ● ● ●

Endotoxin (EU/ml) ● ● ● ● ● ● ● ●

Hemoglobin (mg/dl) ● ● ● ● ● ● ● ● ● ● ● ● ●

Mycoplasmas ● ● ● ● ● ● ● ● ● ● ● ● ●

Osmolality ● ● ● ● ● ● ● ● ● ● ●

pH ● ● ● ● ● ● ● ● ● ● ●

Sp2 performance assay ● ●

Sterility testing (bacteria/fungi) ● ● ● ● ● ● ● ● ● ● ● ● ●

Total protein ● ● ● ● ● ● ● ● ● ● ● ● ●

VERO performance assay ● ● ● ● ● ●

Viral testing

Bluetongue virus ● ● ● ● ● ●

Bovine adenovirus FA ● ● ● ● ● ●

Bovine parvovirus FA ● ● ● ● ● ●

Bovine respiratory syncytial virus FA ● ● ● ● ● ●

Bovine viral diarrhea virus FA ● ● ● ● ● ● ● ● ●

Cytopathic agents ● ● ● ● ● ● ● ● ●

EIA coggins ● ●

Equine herpes virus ● ●

Equine viral arteritis ● ●

Hemadsorbing agents ● ● ● ● ● ● ● ● ●

Porcine adenovirus ●

Porcine hemagglutinating encephalitis ●

Porcine parvovirus ●

Rabies FA ● ● ● ● ● ● ● ● ●

Reovirus FA ● ● ● ● ● ● ● ● ●

Transmissible gastroenteritis virus ●


Animal Sera



Other Sera Quality Control Test Comparison Base cat. no. 16010 26010 16170 26170 16030 10371 16050 26050 16110 16210 16070 26250 16120

Newborn Calf SerumNewborn Calf Serum,

heat-inactivated Bovine SerumBovine Serum

heat-inactivated Donor Bovine SerumDonor Bovine Serum

with iron Horse SerumHorse Serum,

heat-inactivated Chicken Serum Goat Serum Lamb Serum Porcine Serum Rabbit Serum

Cytotoxicity assay ● ● ●

Electrophoretic pattern ● ● ● ● ● ●√ ● ● ● ● ● ● ●

Endotoxin (EU/ml) ● ● ● ● ● ● ● ●

Hemoglobin (mg/dl) ● ● ● ● ● ● ● ● ● ● ● ● ●

Mycoplasmas ● ● ● ● ● ● ● ● ● ● ● ● ●

Osmolality ● ● ● ● ● ● ● ● ● ● ●

pH ● ● ● ● ● ● ● ● ● ● ●

Sp2 performance assay ● ●

Sterility testing (bacteria/fungi) ● ● ● ● ● ● ● ● ● ● ● ● ●

Total protein ● ● ● ● ● ● ● ● ● ● ● ● ●

VERO performance assay ● ● ● ● ● ●

Viral testing

Bluetongue virus ● ● ● ● ● ●

Bovine adenovirus FA ● ● ● ● ● ●

Bovine parvovirus FA ● ● ● ● ● ●

Bovine respiratory syncytial virus FA ● ● ● ● ● ●

Bovine viral diarrhea virus FA ● ● ● ● ● ● ● ● ●

Cytopathic agents ● ● ● ● ● ● ● ● ●

EIA coggins ● ●

Equine herpes virus ● ●

Equine viral arteritis ● ●

Hemadsorbing agents ● ● ● ● ● ● ● ● ●

Porcine adenovirus ●

Porcine hemagglutinating encephalitis ●

Porcine parvovirus ●

Rabies FA ● ● ● ● ● ● ● ● ●

Reovirus FA ● ● ● ● ● ● ● ● ●

Transmissible gastroenteritis virus ●


196


Final Product Quality ControlA representative number of quality control samples from each production lot are analyzed using the following methods:Chemical checks. Osmolality is measured using freezing point depression to verify compliance with product specifications. All osmometers are calibrated daily, using standard solutions. pH is measured to verify compliance with product specifications. All pH meters are calibrated daily using standard solutions.

Electrophoretic profiles are monitored to confirm serum identity and integrity. Samples are applied to a cellulose acetate medium and allowed to migrate in a buffer system. The strips are stained, cleared, dried, and scanned using a densitometer to determine relative concentrations of albumin and globulin fractions.

To verify that proper collection and processing procedures have been used, hemoglobin is measured spectrophotometrically using a modified Fleming method 1 for oxyhemoglobin determination.

As the age of the animal increases, the total protein increases proportionately. To confirm animal age and verify compliance with product specifications, total serum protein is measured using an automated Biuret method. The absorbance of a sample combined with the Biuret reagent is measured at various wavelengths using a colorimeter. Results are automatically calculated and compared to a standard curve, and protein values are calculated in grams per decilitre.Stability testing program. The expiration date shown on every in vitro diagnostic (IVD)-labeled product is an indication of how long the product will deliver effective performance. Our stability program establishes and verifies product expiration dates. Product groups are monitored at periodic intervals to determine the length of time that the product will deliver acceptable performance under recommended storage conditions.Microbiological tests. The absence of bacterial and fungal contamination is confirmed using methods referenced in the current edition of the U.S. Pharmacopeia (USP) and/or the European Pharmacopoeia.

All sera are tested for the absence of mycoplasmas using the large-volume method of Barile and Kern.2 The test is accurate within the detection limits of the methods referenced. The samples are pooled and cultured in broth for a minimum of three weeks, with no fewer than four subcultures onto mycoplasma agar plates. The plates are incubated at 36°C ± 2°C under both aerobic and anaerobic (95% nitrogen, 5% CO2) con di tions. The agar plates are examined microscopically for growth at weekly intervals during the test procedure. Dienes stain is used to detect myco plasma colonies in agar plates that are suspected of contamination. The plates then are reexamined microscopically. Inoculated broth bottles are periodically monitored for turbidity and evidence of pH shift. All bovine sera of Australian , New Zealand, and USA Origins are screened for noncultivatable mycoplasmas using the Hoechst fluorescent DNA stain 3 to detect the presence of mycoplasmas that are noncultivatable by the broth method, including species of M. hyorhinis, within the detection limits of the test method referenced.Viral tests. Adventitious agents are detected using procedures from the Code of Federal Regulations, Title 9 CFR, Part 113.53, “Requirements for Ingredients of Animal Origin”.

Bovine cell cultures previously demonstrated to be free of known adventitious agents are passaged for three subcultures in growth medium containing 15% test serum for 21 days. Negative reference cultures are maintained in parallel, using control medium and serum previously demonstrated as negative for adventitious agents. All cultures are examined throughout this period for evidence of virus-induced morphological changes or cytopathogenic effects. At day 14 of a 21-day culture period, parallel culture flasks containing test serum are evaluated by standard virus-testing protocols, as follows.A flask of test cells is trypsinized, and cells are added to six chamber slides, with a total surface area of 6 cm2. Negative reference slides are prepared in parallel. Individual positive controls are prepared by adding strains of bovine viral diarrhea virus (BVDV), and for certain sera as detailed in the Quality Control Text Comparison, bovine parvovirus, bovine adenovirus, rabies virus, and reovirus to individual chamber slides of test cultures. After an additional seven days of culture (21 days total), viruses are detected by fluorescent antibody techniques.

Cytopathogenic effects (CPE) or other virus-induced effects are observed through hematoxylin and eosin staining of test cultures. Cells are examined for presence of CPE, inclusion bodies, syncytia formation, or other abnormal effects.

Cells from a second flask are tested for hemadsorption (HAD) of human type “O,” guinea pig, and day-old chick erythrocytes, following a procedure patterned after 9 CFR 113.46(b). The test is conducted at 2°C to 8°C and 20°C to 24°C. Negative control cells previously infected with PI-3 virus serve as positive controls. Uninfected cells are used as the negative control.

These tests detect the presence of viral contamination within the detection limits of the methods referenced. With the exception of non-cytopathic BVDV in Fetal Bovine Serum,cell batches of serum must test negative for virus before release for sale.Endotoxin test. The Limulus Amoebocyte Lysate (LAL) gel-clotting procedure is used to measure endotoxin values in all FBS 1 with the exception of FBS and other blood and sera products manufactured in New Zealand.

New Zealand uses a chromogenic kinetic test procedure which measures the concentration of endotoxin spectrophotometrically, based on the initial rate of reaction.4

References1. Fleming, A.F. and Woolf, A.J. (1965) Clin. Chem. 12, 67.

2. Barile, M.F. and Kern, J. (1971) Proc. Soc. Exp. Biol. Med. 138, 432.

3. Chen, J.R. (1977) Exp. Cell Res. 104, 255.

4. Current USP.


Animal Sera



Performance Tests: Fetal Bovine Sera (FBS)The most important criterion in selecting FBS is its ability to support the growth of particular cell types. Therefore, in addition to ensuring that each lot of FBS passes our stringent sterility control tests, we simulate laboratory conditions to test each lot for three important performance character istics: cloning efficiency, plating efficiency, and diploid fibro blast growth promotion.1 Our exclusive performance testing procedures provide quality sera that are defined in terms of their suitability for these three applications. These tests help ensure that each lot of FBS we select for you will provide optimal results in your application.

Cloning efficiency assay. Cat No`s 10106, 10108, 10109, 10270, 10500The cloning efficiency assay analyses the ability of each batch to support cloning and growth of myeloma cells and derived hybridomas. Cells used to determine cloning efficiency are Sp2/O-Ag14 or P3x63 Ag8.653. These are routinely maintained at log phase RPMI 1640 Medium supplemented with 10% Foetal Bovine Serum. The Serum is assayed in microtitre plates and the demanding one cell per well test simulates laboratory conditions for selection of a single hybrid. Cloing assay results are normalised relative to an established reference serum.

The cell suspension being used is adjusted to the desired concentra-tion of 5 cells/ml by dilution with RPMI 1640 Medium supplemented with 20% test or reference serum.

The cell suspensions are then swirled gently to evenly distribute the cells before dispensing 200 μl into each well of a 96 well microtitre plate thus giving a final concentration of 1 cell per well.

Cloning efficiency is calculated as follows:

cloning efficiency = average number of positive wells

× 100 total number of wells inoculated

The relative cloning efficiency (RCE) as follows:

RCE = cloning efficiency of test serum

cloning efficiency of reference serum

Cloning Efficiency Assay. Cat No`s. 16000, 10082, 26140, 16140, 10099, 10100The cloning efficiency assay analyses the ability of each FBS lot to sup-port cloning and growth of murine The following cell lines are validated for use in this assay:• Sp2/O-Ag14(ATCC#CRL1581)• P3x63-Ag8.653(ATCC#CRL1580)

Each FBS lot is assayed in duplicate microtitre plates at two serum concentrations and at two cell innocula (10% serum and one cell/well 4% serum and 5 cells/well). Cloning assay results are normailsed relative to an established reference serum. The higher serum concentration is representative of many routine procedures in hybrid selection, while the lower serum concentration is a more stringent test designed to augment detection of subtle variations among serum lots. The remaining 1-cell/well test simulates laboratory conditions for selection of a single hybrid. The certificate of analysis, provided with each lot of FBS, reports cloning-efficiency as an average of the two test conditions.

Plating Efficiency AssayCat No`s. 10106, 10108, 10109, 10270, 10500The plating efficiency assay determines the ability of a particular batch of FBS to grow a continuous adherent cell line from a very low inoculum density. BHK-21 cells are selected for use in the assay because of their ability to detect subtle variations in serum ability to support cellular attachment and proliferation.

BHK-21 cells are maintained in logarithmic phase at + 37oC in Glasgow MEM supplemented with 10% Newborn Calf serum and 10% Trytose Phosphate Broth. Nutrient Mix F12 (HAM) supplemented with 1% glutamine is used as growth medium.

Five flasks of growth medium supplemented with 20% test serum are inoculated at 40 cells/ml. After incubation for 7 days at + 37OC and 5% CO2, in a humidified incubator, the cells in the flasks are stained with Giemsa and the cell colonies counted. Results are normalised relative to a previously qualified reference serum as follows:

Plating Efficiency

PE = no of Colonies Counted

× 100 No of Cells Inoculated

Relative Plating Efficiency (RPE) is calculated as:

RPE = PE in Test Serum

PE in control serum

Plating Efficiency AssayCat No`s. 16000, 10082, 26140, 16140, 10099, 10100 The plating efficiency assay uses a human transformed cell line to determine the suitability of each lot for growing continuous adherent cell lines. The cell line validated for determining plating efficiency, A549 (HumanLungCarcinoma,ATCC#CCL,185),wasselectedbecauseofit`sability to support cellular attachment and proliferation.

The A549 cell line is maintained at 36oC ± 2o C at subconfluent density in Dulbecco`s Eagle Medium (D-MEM) supplemented with 10% FBS.

Each FBS lot is tested in triplicate at two serum concentrations and at two cellular inocula (10% serum and 100 cells/well, 4% serum and 200 cells/well. After incubation for approximately 10 to 14 days at + 36oC ± 2o C in a humidified CO2 incubator, the plating efficiency is determined by counting cell colonies. Results are normalized relative to the reference control serum. The two serum concentrations demonstrate the lot`s ability to support growth at both reduced and standard levels. The certificate of analysis, provided with each lot of FBS, reports plating efficiency values as an average of the last two test conditions.


198


Diploid fibroblast growth promotion assay. The growth promotion assay measures the ability of each FBS lot to support proliferation of fastidious human diploid fibroblasts through multiple subcultures. The WI-38 cell line (humandiploid lungfibroblast,ATCC#CCL75) is validated forusein this assay.

WI-38 cells are inoculated into duplicate 25 cm2 flasks at low density (2 × 105/flask) with 5% FBS and propagated through three sequential 7-day subcultures. At each passage, the cells are subcultured to the original cell inoculation density. The stressful split ratio, number of population doublings, and reduced serum concentration are stringent measurements of the serum lot’s ability to grow fastidious diploid cells. The three sequential subcultures ensure a true measurement of the test lot’s growth- promoting ability by minimizing any residual effects of the previous growth medium. The Certificate of Analysis provided with each lot of FBS reports the average number of cells per duplicate flask from the third subculture. Test values are normalized relative to the reference control.Diploid fibroblast growth assay methodology. The growth promotion assay is performed as follows:1. Growth medium supplemented with 5% test serum or a previously

qualified reference serum is prepared. The basal growth medium is HBME:EBME (1:1) containing L-glutamine. Fresh 5% serum- supplemented growth medium is prepared for each fluid change and subculture during the assay, as required.

2. WI-38 cells (2 × 105) at a low passage level are inoculated into each flask containing 9.5 ml of the appropriate growth medium in duplicate 25 cm2 flasks. The flasks are maintained loosely capped in a 4–6% CO2, 36°C ± 2°C atmosphere for the initial 24 hours. The flasks then are capped tightly and incubated for 7 days at 36°C ± 2°C, with complete medium replenishment at day 2 to day 3.

3. Cells in each of the duplicate flasks per reference or test serum are quantitatively harvested by trypsinization at day 7, then pooled and counted. Cells are subcultured by inoculating 2 × 105 WI-38 cells into new duplicate 25 cm2 flasks containing fresh growth medium that has been supplemented with the same lot of reference or test FBS used for the previous passage. The flasks are equilibrated and incubated as described in step 2.

4. The assay is continued as described through three passages, and the average number of cells/flask for each reference or test serum is calculated at the end of each passage. The relative growth rate (RGR) in each test serum during the final subculture is expressed as a fraction of the cell growth obtained in the reference serum:

% RGR = average cells/flask in test serum

average cells/flask in reference serum

ReferenceMartinez, E.C., Cady, D.J., Grefrath, P.L., and Jayme, D.W. (1985) In Vitro 21, 30A.

Sf9 cell growth promotion assay. The insect assay assures that the FBS lot you purchase will support the growth of Sf9 cells. Upon receipt, all you have to do is heat-inactivate the serum for 30 min at 56°C with mixing. This assay protocol can also be used for the biological performance assay of lactalbumin hydrolysate, yeastolate, and other growth supplements tested as raw materials. The accuracy of this assay is dependent on adherence to a routine cell maintenance schedule. Cell viability should be at least 80% as determined by trypan blue dye exclusion.

This assay is performed on lots of certified FBS (base cat. nos. 16000, and 10082) and on selected lots of qualified FBS (base cat. nos. 26140 and 10099).

Sf9 cell growth assay methodology1. Complete test media are prepared using a previously approved lot

of Grace’s Insect Culture Medium supplemented with 10% test or reference heat-inactivated FBS.

2. Stock Sf9 cells are dispensed into 50 ml centrifuge tubes and centri fuged at 50 × g for 5 min. Each tube is resuspended with complete medium containing test or reference heat-inactivated FBS at 10% concentration.

3. After inverting the tubes several times, a 1 ml sample is removed from each tube. A viable cell count is performed using trypan blue dye exclusion.

4. If viability is ≥80%, the tube is inverted several times and a 0.25 ml sample is removed from the tube. A cell count is then performed using a Coulter counter.

5. Cells are inoculated into Erlenmeyer flasks at a final concen tration of 2.75 × 105 cells/ml. After inoculation, another cell count is performed to assure a cell density of 2.5 to 3.0 × 105 cells/ml.

6. The flasks are incubated on a shaker apparatus at 27°C ± 1°C at 80–90 rpm for 96 hours.

7. Two 0.25 ml samples are removed from each flask and counted on the Coulter counter. A sample is also removed for determination of cell viability.

8. The relative cell number (RCN) is determined by comparing the test cell density to the control cell density.


Animal Sera



Additional Tests: Certified (US)The following additional tests are performed on base cat. nos. 16000 and 10082. You will receive a Certificate of Analysis documenting the results of each quality control assay.Bacteriophage tests.2 Testing for the presence of bacteriophages using a plaque-assay method.3 A representative sample from each serum lot is added to tryptose phosphate agar containing a suspension of bacterio-phage-sensitive E. coli (C-3000 or K-12). The mixture is then layered onto tryptose phosphate agar plates and incubated at 36°C ± 2°C for approximately 24 hours, and the plates are observed for plaque formation.

Biochemical Profile Testsalkaline phosphatasebicarbonatebilirubin (total)blood urea nitrogen (BUN)BUN/creatinine ratio calciumchloridecholesterolcreatininegamma glutamyl transpeptidase (GGTP)glucose Sera Tested for Cytotoxicity

Product Base cat. no.

Chicken Serum 16110

Lamb Serum 16070

Porcine Serum 26250

Hormone Profile Testsestradiol testosterone insulin thyroxine (T4) progesterone

Hemoglobin TestAll lots of US Certified FBS are guaranteed to be ≤ 15 mg/dl hemoglobin with ≥70% comprised of oxyhemoglobin.

Additional Tests: ES Cell-Qualified FBSGibco® ES Cell-Qualified FBS is specially tested for the ability to sustain the undifferentiated cellular morphology of embryonic stem cells.

high-density lipoprotein (HDL)lactate dehydrogenase (LDH)low-density lipoprotein (LDL)potassium

phosphorous (inorganic)serum glutamyl oxaloacetate transaminase (SGOT)sodiumtotal iron binding capacitytransferrintriglycerides (TG)uric acid

Performance Tests: Other SeraBovine sera and Newborn Calf sera. Each lot of Bovine Serum and Newborn Calf Serum (see Other Sera Ordering Information, pages 126–128) is tested for its ability to support the growth of Vero cells over three subcultures. At each passage, the cells are subcultured to the original cell-inoculation density. Results are compared to parallel results obtained using control growth medium containing a previously characterized reference serum.Horse serum. Each lot of Horse Serum (see Other Sera Quality Control Tests, pages 132–133) is tested for its ability to support the growth of Sp2/O-Ag14 (murine myeloma) cells in control medium containing the test lot of serum. Results are compared to parallel results obtained using control growth medium containing a previously characterized reference serum.

Cytotoxicity Tests: Other SeraThe performance testing procedures described in the preceding section are, by design, stringent tests for cytotoxicity as well. For some sera that do not undergo performance testing, we test specifically for cytotoxicity. These products are listed in the table below. Test samples are examined for the ability to support the attachment and proliferation of three adherent cell lines. The test and reference cultures are monitored for evidence of nutritional deficiency, abnormal morphology, or cytotoxicity.


200


Special Processing of SeraDialysed SeraFor researchers requiring dialysed sera, we provide sera that are dialysed against 0.15 M NaCl using 10,000 molecular weight cut-off membrane until the glucose level is <5.0 mg/dl, as determined by the Glucose oxidase/peroxidase (GOD/POD) method (1, 2). The tangential flow filtration (TFF) process allows serum to be processed and final filtered on the same day. To prevent precipitation and inactivation of serum peptides, we do not perform exhaustive dialysis. Thus, we do not certify that all low molecular weight dialyzable components, such as amino acids, are totally removed by our processing.

References1. Keston, A. (abstract) 129th meeting of ACS, September 1956, 31C

2. Trinder, P (1969) Ann. Clin. Biochem. 6.24

Gamma Irradiated SerumSerum is one of the most commonly used supplements in many cell culture systems and as any biological derived product, it carries the risk of being contaminated by adventitious agents, particularly viruses and mycoplasma. Although mycoplasma can be successfully removed by filtration through 0.1 * pore size filter, the same is not true for most viruses. Various methods for eliminating virus contamination have been applied to serum. Gamma irradiation is the traditional method of choice for inactivating virus in serum as it enables effective virus inactivation over a wide spectrum of virus types, unlike UV irradiation.

Heat Inactivated SeraWe heat inactivate serum by mixing it in a water bath at a thermostatically controlled temperature of + 56o C for 30 mins.

Ultra Low lgG FBSThe advent of the Biotechnology industry and the production of “biologicals” from Cell Culture has highlighted some disadvantages in using Foetal Bovine Serum in certain production regimes. For example, the presence of immunoglobulins (in particular IgG) can cause difficulties in the purification of certain biologicals (e.g. Monoclonal Antibodies). We have developed a proprietary chromatographic procedure for the removal of gamma globulin from FBS. This procedure allows us to produce FBS with very low concentrations of IgG (<5mg/ml) while maintaining full biological activity in the sera. Afurther significant feature of serum produced using this process is the extremely low BVD antibody titre (Serum Index < 1).

Charcoal Stripped FBSFor researchers who require lower levels of hormones, especially estradiol, we offer this special FBS that has been processed using a proprietary charcoal/dextran treatment to reduce hormone and growth factor levels.

Virus inactivation under experimental conditions (minimum dose of - 25kGy)

Virus

Spiked serum before

treatment (pfu/ml)

Spiked serum after

gamma irradiation

Reduction index

for total process

BVD 8.7 x 104 < 0.1 > 8.7 x 105

IBRV 1.6 x 105 < 0.1 > 1.6 x 106

P13 2.0 x 105 < 0.1 > 2.0 x 106

PPV 2.0 x 105* < 0.1* > 2.0 x 106

RE03 1.8 x 105 < 0.1 > 1.8 x 106

* Titre expressed as TCID50 values


Animal Sera



Notes:


202


Notes:


Appendix IGibco® Universal Bags . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 204

Prefilled Wave Bag . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 207

new Lab Armortm Beads . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 208

PD-Directtm Bioprocess Services . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 209

Cell Culture basics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 210

new Supply Centre by Life Technologiestm . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 213

Formulas and Formula Weights for Commonly Used Medium Components . . . . . . . . . . . . . . . . . 216

Troubleshooting: Cell Culture . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 218

Useful Numbers for Cell Culture . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 219

Buffer Systems for Cell Culture . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 219

Specialty Media and Applications . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 219

Serum-free Media and Applications . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 220

Hybridoma Culture. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 220

Chinese Hamster Ovary (CHO) Cell Culture. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 221

Human Embryonic Kidney (293) Cell Culture . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 221

Mammalian Cell Culture for Virus Production . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 221

Insect Cell Culture . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 222

Neuronal Cell Culture . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 222

Blood and Bone Marrow Culture . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 223

Stem Cell Culture . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 223

Other Mammalian Cell Culture . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 223

Adaptation of Cell Cultures to a Serum-free Medium. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 224

Adaptation of Cell Cultures to an Alternative Serum . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 225

Adaptation of Cells from Monolayer to Suspension Culture . . . . . . . . . . . . . . . . . . . . . . . . . . . 225

Stability of GlutaMAX™-I vs. L-Glutamine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 226

Cryopreservation of Mammalian Cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 227

Thawing of Cryopreserved Cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 227

Dissociation of Cells from Primary Tissue . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 228

Dissociation of Cells from Culture Vessels . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 229

Decontaminating Cultures with Antibiotics and Antimycotics . . . . . . . . . . . . . . . . . . . . . . . . . . 231

Use of Antibiotics and Antimycotics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 231

Conversion Factors, Units, and Abbreviations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 232

Genetic Code and Amino Acid Abbreviations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 233

Appendix I Reference Guide

204


Gibco® media and reagents —available in flexible packaging

Specifically designed to increase productivity and reduce costs ■

Conveniently prefilled in a variety of formulations and sizes ■

Easily integrated with most systems—unique universal design ■

provides multiple connection optionsFor over 49 years, gibco® has built a reputation for innovation and service, with a commit-ment to quality. Our focus on packaging is no exception and our engineers have worked closely with industry leaders to extend the range of liquid delivery systems. Gibco® universal bags are designed with common tubing connections for easy and con-venient integration into all your media applications. You can order your media prefilled in a 5, 10, or 20 L universal bag (Figure 1) and choose from a variety of stocked formulations.If your project requires a media and bag not offered prefilled, you can contact a knowl-edgeable member of Invitrogen’s cell culture custom team to assist you with selecting the right configuration. Packaging options are offered in a consistent film ranging from 250 mL up to 1,000 L.

Superior-quality filmThe gibco® universal bag incorporates an animal origin–free 9101 film with an ultra low-density polyethylene media contact layer (Figure 2). It has been fully qualified* and widely used in many cell culture applications.

Consistent film across gibco®’s bag offering makes scale up easier, ■

eliminating the need for multiple validations. All bag sizes, ranging from 250 mL to 1,000 L, are manu-factured using the 9101 film Extensive documentation for the 9101 film is available and includes: ■

USP Class VI, leachables, extractables, gas permeability, and biological and physical propertiesSuperior gas transmission rates improve product consistency ■

(Figure 4Transparent film allows clear and improved view of bag contents ■

(Figure 3)Proven stability for pH and cell performance ensure product quality ■

(Figure Y with 2 graphs to be provided).

Figure 1. Gibco® universal bags have a f lexible four-por t design offering versatility and convenience. (1) female-threaded luer; (2) blood spike; (3) male-threaded luer; (4) MPC quick connect.

Figure 3. New 9101 film (on left) provides superb clarity of contents

Figure 2. 9101 film construction details.

1. 50 µm Polyethylen

2. 10 µm Adhesive layer 3. 20 µm Ethyl Vinyl Alcohol (Gas barrier

4. 10 µm Adhesive Layer5. 230 µm Ultra low-density polyethylene (Contact layer).


Flexible, four-port designThe gibco® line of flexible packaging allows you to utilize and integrate a single-use, dis-posable container with a variety of automated cell culture instruments, disposable bioreac-tor systems, and sterile connection processes without the need for costly customization. Gibco® universal bags are compatible with four different mechanical connection methods (blood spike, male-threaded luer, female-threaded luer, and MPC quick connect (Figure 5); featuring:

Versatile and closed media transfer system utilizing three ■

preconnected C-Flex® tub-ing transfer linesVariety of tubing sizes for flow rate flexibility (ID × OD):• 1/8” × 1/4” ■

(3.2 mm x 6.4 mm)• 3/16” × 5/16” (4.8 mm x 7.9 mm)• 3/8” × 5/8” (9.5 mm x 15.9 mm) ■

Compatibility with the most commonly used thermoplastic tubing ■

Improved packagingGibco® universal bags are shipped in a reinforced, 100% recyclable container. Made from heavyweight corrugate material, these boxes provide a solid system to transport and store your gibco® universal bags. Durable five-layer handles provide a strong hold to comfortably and confidently carry your media.

Figure 4. Gas transmission rates. The gibco® universal bag shows superior gas transmission rates in compari-son to current bags on the market.

Figure 6. Gibco universal bags are ideal for many cell culture applications.

Figure 5. Four-port design. Gibco® universal bags have a flexible four-port design offering versatility and convenience: (1) female-threaded luer, (2) blood spike, (3) male-threaded luer, and (4) MPC quick connect.


206


Order gibco® media and reagents in universal bags from Life Technologiestm today.Product Quantity Cat. No.

Balanced salts

DPBS without calcium chloride, magnesium chloride, or phenol red 5 L 10 L 20 L

14190 -342 14190-359 14190-367

PBS without phenol red, pH 7.4 5 L 10 L

10010-002 10010-001

HBSS without calcium chloride, magnesium chloride, or magnesium sulfate† 5 L 10 L

14170-195 14170-203

Basal media

DMEM high glucose with L-glutamine, without sodium pyruvate 5 L 10 L

11965-167 11965-175

DMEM high glucose with GlutaMAX™-I, without sodium pyruvate† 5 L 10 L

10566-032 10566-040

DMEM high glucose, with L-glutamine and sodium pyruvate 5 L 10 L

11995-115 11995-123

DMEM high glucose, with GlutaMAX™-I and sodium pyruvate 5 L 10 L

10569-069 10569-077

DMEM with L-glutamine and HEPES buffer, without sodium pyruvate 5 L 10 L

12430-112 12430-120

DMEM with GlutaMAX™-I and HEPES buffer, without sodium pyruvate† 5 L 10 L

10564-037 10564-045

DMEM/F-12 with L-glutamine and HEPES buffer 5 L 10 L

11330-099 11330-107

RPMI Medium 1640 with L-glutamine 5 L 10 L

11875-168 11875-176

RPMI Medium 1640 with GlutaMAX™-I 5 L 10 L

61870-143 61870-150

RPMI Medium 1640 with GlutaMAX(TM)-I and HEPES buffer 5 L 10 L

72400-146 72400-153

RPMI Medium 1640 with L-glutamine and HEPES buffer 5 L 10 L

22400-147 22400-154

Reagents

Distilled Water 5 L 10 L 20 L

15230-25315230-26115230-279

TrypLE™ Express 1X with phenol red 5 L 12605-093

TrypLE™ Express 1X without phenol red 5 L 12604-054

Expression Media

Sf-900™ II with L-glutamine 5 L 10 L 20 L

10902-161 10902-179 10902-187

Sf-900™ III with L-glutamine 10 L 20 L

12658-035 12658-001

CD CHO 10 L 20 L

10743-001 10743-002

FreeStyle™ 293 10 L 20 L

12338-001 12338-002

For additional information, to place your order, or to request a custom format, formulation, or film construction, please contact Life Technologiestm Customer Service or visit www.invitrogen.com/universalbags.

† Make to stock product.

Gibco® Universal Bags

gibco® cell culture catalogueAppendix I



Prefilled Wave BagThe Cellbag® media bag from Wave Biotech is now available prefilled with gibco® media

Ready to ship—media formulations include Sf-900™ II and FreeStyle™ 293 ■

Convenient—Cellbag® chambers are filled and ready to use ■

Customizable—you choose the gibco® medium formulation and volume ■


Sf-900™ II SFM 10 L fill in 20 L Wave Cellbag® 0070037BA

FreeStyle™ 293 Expression Medium 7 L fill in 20 L Wave Cellbag® 0070049BA 8 L fill in 20 L Wave Cellbag® 0070049BB


208


Lab Armor™ BeadsReplace the water in your lab bath to help prevent contaminationStanding, warm water in your laboratory water bath can introduce unknown bacteria, yeasts, fungi, mycoplasmas, and chemical contaminants into your lab. These contaminants can affect your cell culture reagents, resulting in distorted data and unpredictable outcomes.

Lab Armor™ Beads replace the water in your laboratory water bath and reduce the potential for contamination of your media, reagents, and cells. With Lab Armor™ Beads, you can protect your research and use your lab bath with confidence.

Confidence and reproducibility with Lab Armor™ Beads and gibco® productsGibco® cell culture products deliver reproducible results and award-winning service while helping you reduce the risk of contamination in your lab. Use gibco® cell culture products and Lab Armor™ Beads together every time you perform cell culture. Remember to include Lab Armor™ Beads in your next order of gibco® cell culture products.

Gibco® cell culture products and Lab Armor™ Beads help you achieve reproducible results every day.Learn more at www.invitrogen.com/labarmorbeads.

Lab Armor™ Beads are low-maintenance and highly consistent

Clean—water baths must be routinely monitored, cleaned, and ■

maintained; Lab Armor™ Beads only need a light spray of biodegradable disinfectant every 2–4 weeksConvenient—no need to plan around warm-up times, or worry about ■

burnout when water evaporatesOrganized—Lab Armor™ Beads naturally hold items in place, no matter ■

the size or shape; racks, floats, and weights are not requiredConsistent—greater temperature uniformity and maintenance than ■

water

Lab Armor™ Beads are the environmentally responsible choice

Eliminates biocides—liquid disinfectants used for water bath ■

maintenance are harmful to the environment; periodic use of a biodegradable disinfectant like ethanol is all that is required to keep Lab Armor™ Beads free of contaminationRecyclable—formed from ultrapure, recyclable material ■

Energy-efficient—set at 35°C, Lab Armor™ Beads consume less than ■

one half of the energy required by water




PD-Direct™ Bioprocess ServicesInvitrogen has over 49 years of experience in the cell culture business and over this time, the Life Technologiestm name and reputation has stood as the recognized leader in cell culture systems. Life Technologiestm expanded on this tradition in innovative cell culture products through the creation of a team focused on process development, PD-Direct™ Bioprocess Services. The PD-Direct™ team responds to core customer needs by offering customers comprehensive service from cell line and medium development to process optimization (Figure 1).

Upstream Optimization ServicesThe PD-Direct™ team brings together the best scientists and technologies in the area of upstream optimization. Through a proven approach of applying high-throughput technologies to process design within an integrated workflow, our systematic approach yields performance improvements in your manufacturing process. The PD-Direct™ upstream services range from clone-specific process and media optimization to platform development bridging Cell line development, cloning, basal media, and feed supplements. Our ability to bring this step change in performance comes from different elements:

Unrivaled breadth of historical experience in media screening and ■

media formulation across cell lines and cell typesA high-throughput workflow integrating multi-factor statistical ■

experimental design with Simcell miniturised bioreactors and Hamilton Starplus automated media formulation.Comprehensive media analytics to provide vital in-process ■

information Component harmonization and elimination of unnecessary or inhibitory ■

components to reduce system complexityExpertise from cloning through large-scale production that empowers ■

integrated media platforms that minimize adaptation time and effort

Cell Line Services The science of cell line development has become complex. Increasing production demands with shorter development timelines require a breadth of investments in platforms, technologies, and personnel. Factors such as transfection efficiency, cell productivity, media conditions, and cell line stability must be incorporated into an overall program of cell line optimization. From a myriad of expression enhancers to high-throughput screening, the challenges of optimization continue to grow. Additionally, intellectual property, technology harmonization, and final product quality are other considerations that complicate the process.

In response, Life Technologiestm has integrated an array of services to simplify cell line development, through PD-Direct™ Bioprocess Services. We offer you a suite of the best cell line development technologies from across the industry and our in-depth knowledge. This unique combination will yield a high-quality production cell line that will serve as the founda-tion for your therapeutic product development for years to come.

Our cell line services include:Parental cell line optimization through natural adaptation or accelerated ■

evolution utilizing the Revolution™ technologyPreoptimized, serum-free FreeStyle™ expression services to produce a ■

protein within a week in suspension HEK or 293 culturesCel l l ine engineer ing with propr ietar y molecular biology ■

technologies Recombinant cell line creation with CHO cell lines that incorporates ■

industry-leading and IP-friendly vector componentsClone selection or enrichment through the use of the ClonePix ■ FL high- throughput fluorescent screening technologyExpression enhancement with the Revolution™ technology for difficult- ■

to-produce proteins

Get your process goingThrough a partnership with Invitrogen, you can join industry leaders who have discovered the difference that Invitrogen’s innovation can bring to their projects. Whether a point solution for upstream optimization or cell line creation, or an integrated process development solution, PD-Direct™ Bioprocess Services is the trusted partner that brings the expertise and breadth of Invitrogen to solving your process development needs. Get your process going by visiting us at www.invitrogen.com/pddirect or emailing us at [email protected].

Figure 1—PD-Direct™ Bioprocess Services expands your process development options.


210


Cell Culture basicsThis section provides information on the fundamentals of cell culture, including the selection of the appropriate cell line for your experiments, media requirements for cell culture, adherent versus suspension culture, and morphologies of continuous cell lines available from Life Technologiestm.

Note that the following information is an introduction to the basics of cell culture, and it is intended as a starting point in your investigations. For more in-depth information, we recommend that you consult published literature and books, as well as the manuals and product information sheets provided with the products you are using.

Cell Lines

Selecting the Appropriate Cell LineConsider the following criteria for selecting the appropriate cell line for your experiments—

Species: Non-human and non-primate cell lines usually have fewer ■

biosafety restrictions, but ultimately your experiments will dictate whether to use speciesspecific cultures or not.Functional characteristics: What is the purpose of your experiments? ■

For example, liver- and kidney-derived cell lines may be more suitable for toxicity testing.Finite or continuous: While choosing from finite cell lines may give you ■

more options to express the correct functions, continuous cell lines are often easier to clone and maintain.Normal or transformed: Transformed cell lines usually have an increased ■

growth rate and higher plating efficiency, are continuous, and require less serum in media, but they have undergone a permanent change in their phenotype through a genetic transformation.Growth conditions and characteristics: What are your requirements with ■

respect to growth rate, saturation density, cloning efficiency, and the ability to grow in suspension? For example, to express a recombinant protein in high yields, you might want to choose a cell line with a fast growth rate and an ability to grow in suspension.Other criteria: If you are using a finite cell line, are there sufficient stocks ■

available? Is the cell line well-characterized, or do you have to perform the validation yourself? If you are using an abnormal cell line, do you have an equivalent normal cell line that you can use as a control? Is the cell line stable? If not, how easy it is to clone it and generate sufficient frozen stocks for your experiments?

Acquiring Cell LinesYou may establish your own culture from primary cells, or you may choose to buy established cell cultures from commercial or non-profit suppliers (i.e., cell banks). Reputable suppliers provide high quality cell lines that are carefully tested for their integrity and to ensure that the culture is free from contaminants. We advise against borrowing cultures from other laboratories because they carry a high risk of contamination. Regardless of their source, make sure that all new cell lines are tested for mycoplasma contamination before you begin to use them.

Life Technologiestm offers a variety of primary cultures and established cell lines, reagents, media, sera, and growth factors for your cell culture experiments. The Appendix section contains a list of the more commonly used cell lines available from Life Technologiestm. For more information on Life Technologiestm and gibco® products, refer to www.invitrogen.com.

One of the major advantages of cell culture is the ability to manipulate the physicochemical (i.e., temperature, pH, osmotic pressure, O2 and CO2 tension) and the physiological environment (i.e., hormone and nutrient concentrations) in which the cells propagate. With the exception of tem-perature, the culture environment is controlled by the growth media.

While the physiological environment of the culture is not as well defined as its physicochemical environment, a better understanding of the components of serum, the identification of the growth factors necessary for proliferation, and a better appreciation of the microenvi-ronment of cells in culture (i.e., cell-cell interactions, diffusion of gases, interactions with the matrix) now allow the culture of certain cell lines in serum-free media.

Adherent vs Suspension CultureThere are two basic systems for growing cells in culture, as monolayers on an artificial substrate (i.e., adherent culture) or free-floating in the culture medium (suspension culture). The majority of the cells derived from vertebrates, with the exception of hematopoietic cell lines and a few others, are anchorage-dependent and have to be cultured on a suitable substrate that is specifically treated to allow cell adhesion and spreading (i.e., tissue-culture treated). However, many cell lines can also be adapted for suspension culture. Similarly, most of the commercially available insect cell lines grow well in monolayer or suspension culture. Cells that are cultured in suspension can be maintained in culture flasks that are not tissue-culture treated, but as the culture volume to surface area is increased beyond which adequate gas exchange is hindered (usually 0.2–0.5 mL/cm2), the medium requires agitation. This agitation is usually achieved with a magnetic stirrer or rotating spinner flasks.




MediaThe culture medium is the most important component of the culture environment, because it provides the necessary nutrients, growth factors, and hormones for cell growth, as well as regulating the pH and the osmotic pressure of the culture.

Although initial cell culture experiments were performed using natural media obtained from tissue extracts and body fluids, the need for standardization, media quality, and increased demand led to the development of defined media. The three basic classes of media are basal media, reduced-serum media, and serum-free media, which differ in their requirement for supplementation with serum.

Serum is vitally important as a source of growth and adhesion factors, hormones, lipids and minerals for the culture of cells in basal media. In addition, serum also regulates cell membrane permeability and serves as a carrier for lipids, enzymes, micronutrients, and trace elements into the cell. However, using serum in media has a number of disadvantages including high cost, problems with standardization, specificity, variability, and unwanted effects such as stimulation or inhibition of growth and/or cellular function on certain cell cultures. If the serum is not obtained from reputable source, contamination can also pose a serious threat to suc-cessful cell culture experiments. To address this threat, all Invitrogen and gobco® products, including sera, are tested for contamination and guar-anteed for their quality, safety, consistency, and regulatory compliance.

Basal MediaThe majority of cell lines grow well in basal media, which contain amino acids, vitamins, inorganic salts, and a carbon source such as glucose, but these basal media formulations must be further supplemented with serum.

Reduced-Serum MediaAnother strategy to reduce the undesire d effects of serum in cell culture experiments is to use reduced-serum media. Reduced-serum media are basal media formulations enriched with nutrients and animal-derived factors, which reduce the amount of serum that is needed.

Serum-Free MediaSerum-free media (SFM) circumvents issues with using animal sera by replacing the serum with appropriate nutritional and hormonal formulations. Serum-free media formulations exist for many primary cultures and cell lines, including recombinant protein producing lines of Chinese Hamster Ovary (CHO), various hybridoma cell lines, the insect lines Sf9 and Sf21 (Spodoptera frugiperda), and for cell lines that act as hosts for viral production (e.g., 293, VERO, MDCK, MDBK), and others. One of the major advantages of using serum-free media is the ability to make the medium selective for specific cell types by choosing the appropriate combination of growth factors. The table below lists the advantages and disadvantages of serum-free media.

Adherent Culture Suspension Culture

Appropriate for most cell types, including primary cultures. Appropriate for cells adapted to suspension culture and a few other cell lines that are nonadhesive (e.g., hematopoietic).

Requires periodic passaging, but allows easy visual inspection under inverted microscope.

Easier to passage, but requires daily cell counts and viability determination to follow growth patterns; culture can be diluted to stimulate growth.

Cells are dissociated enzymatically (e.g., TrypLE™ Express, trypsin) or mechanically. Does not require enzymatic or mechanical dissociation.

Growth is limited by surface area, which may limit product yields. Growth is limited by concentration of cells in the medium, which allows easy scale-up.

Requires tissue-culture treated vessel. Can be maintained in culture vessels that are not tissue-culture treated, but requires

agitation (i.e., shaking or stirring) for adequate gas exchange.

Used for cytology, harvesting products continuously, and many research applications.

Used for bulk protein production, batch harvesting, and many research applications.

Advantages Disadvantages

Increased definition Requirement for cell type-specific media formulations

More consistent performance Need for higher degree of reagent purity

Easier purification and downstream processing

Slower growth

Precise evaluation of cellular functions

Increased productivity

Better control over physiological response

Enhanced detection of cellular mediators

Invitrogen offers a wide range of classical basal media, reduced-serum media, and serumfree media, as well as sera, growth factors, supplements, antibiotics, and reagents for your cell culture experiments. The Appendix section contains a list of the more commonly used cell culture products available from Life Technologiestm. For more information on Life Technologiestm and gibco® cell culture products, refer to www.invitrogen.com.


212


pHMost normal mammalian cell lines grow well at pH 7.4, and there is very little variability among different cell strains. However, some transformed cell lines have been shown to grow better at slightly more acidic environments (pH 7.0–7.4), and some normal fibroblast cell lines prefer slightly more basic environments (pH 7.4–7.7). Insect cell lines such as Sf9 and Sf21 grow optimally at pH 6.2.

CO2

The growth medium controls the pH of the culture and buffers the cells in culture against changes in the pH. Usually, this buffering is achieved by including an organic (e.g., HEPES) or CO2-bicarbonate based buffer. Because the pH of the medium is dependent on the delicate balance of dissolved carbon dioxide (CO2) and bicarbonate (HCO3

–), changes in the atmospheric CO2 can alter the pH of the medium. Therefore, it is necessary to use exogenous CO2 when using media buffered with a CO2-bicarbonate based buffer, especially if the cells are cultured in open dishes or transformed cell lines are cultured at high concentrations. While most researchers usually use 5–7% CO2 in air, 4–10% CO2 is common for most cell culture experiments. However, each medium has a recommended CO2 tension and bicarbonate concentration to achieve the correct pH and osmolality; refer to the media manufacturer’s instructions for more information.

TemperatureThe optimal temperature for cell culture largely depends on the body temperature of the host from which the cells were isolated, and to a lesser degree on the anatomical variation in temperature (e.g., temperature of the skin may be lower than the temperature of skeletal muscle). Overheating is a more serious problem than underheating for cell cultures; therefore, often the temperature in the incubator is set slightly lower than the optimal temperature.

Most human and mammalian cell lines are maintained at 36°C to 37°C ■

for optimal growth.Insect cells are cultured at 27°C for optimal growth; they grow more ■

slowly at lower temperatures and at temperatures between 27°C and 30°C. Above 30°C, the viability of insect cells decreases, and the cells do not recover even after they are returned to 27°C.Avian cell lines require 38.5°C for maximum growth. Although these ■

cells can also be maintained at 37°C, they will grow more slowly.Cell lines derived from cold-blooded animals (e.g., amphibians, cold- ■

water fish) tolerate a wide temperature range between 15°C and 26°C.

Note that cell culture conditions vary for each cell type. The consequences of deviating from the culture conditions required for a particular cell type can range from the expression of aberrant phenotypes to a complete failure of the cell culture. We therefore recommend that you familiarize yourself with your cell line of interest, and closely follow the instructions provided with each product you are using in your experiments.




Supply Centre by Life Technologies™ is an electronic, customisable onsite stocking programme offering a premium consignment delivery service.

Right product in the right place at the right time ■

Easy to use ■

Customised to your needs ■

Reduced transaction cost ■

Dedicated support ■

Contact [email protected] for more information.

www.invitrogen.com/supplycentre

Maximise your research time… with 24/7 access to a wide variety of brands

“24/7 access means never having to run out of essential products at that crucial stage in your experiments. Just a phonecall or email to the dedicated Supply Centre team results in queries being handled efficiently and always in a friendly manner.”

Lizzie GaultUniversity of Glasgow, UK

“With the pressure to publish and difficulty to acquire funding for academic research, Supply Centre by Life technologies™ has saved us a great amount of cost and administration time. Working without the Supply Centre is no longer an option.”

Dr. Bart VanaudenaerdeKU Leuven, Belgium


214


Introducing Supply Centre by Life Technologies™

Supply Centre by Life Technologies™. It’s what you need. Now.Life Technologiestm is committed to creating an environment in which the biomedical sciences flourish. Our efforts are focused on a simple goal; how to better meet the needs of scientists. To this end, we have designed our Supply Centre programme for the efficient and convenient storage of products at our customers’ sites, because researchers should not have to put their work on hold while a critical enzyme, reagent, or kit is being shipped.

Convenience – Instant access to staple research products ■

Total Workflow Solution - Customise product mix to best suit your ■

Research requirementsCost effective – Reduce transaction costs, consolidate shipping and ■

streamline invoice processingFlexibility – Ability to order non-stock products ■

Zero risk – All appliances and stock owned & maintained by Life ■

Technologies

The Supply Centre Management System (SCMS) The Supply Centre Management System is a purpose built, web-based interface that allows you to run your Supply Centre easily and efficiently. This flexible system is designed to support your existing business rules, to integrate with your existing procurement platform, and to simplify ordering. The Supply Centre Management System allows full visibility of order history, current stock list, account specific pricing and provides access to administrative reports.




A fully customised solution Each installation is fully customised to suit the customer’s individual requirements with regard to:

Product Mix - Select from the broad portfolio of Life Technologies ■

brands.Location - May be located in either a Lab, Stores area or public area ■

Appliance(s) - Choice of appliance based on product mix and/or ■

selected site or location. Delivery options - Weekly, Bi-Weekly or Monthly, delivery day selected ■

by the customerInvoicing Options - Choice of Standard or Consolidated invoicing ■

Integration with existing procurement solutions ■

Seamless integration with your existing procurement system We recognise that many customers have procurement workflows within their organisations and to support eBusiness and efficiency, purchasing is often carried out within these environments. To support these customer requirements Life Technologiestm can offer a full suite of integration capabilities including:

B2B integration to SCMS (Supply Centre Management System) - ■

Punchout (OCI/Roundtripping)B2B integration to catalogue-based systems ■

These options are provided at no cost to our customers through our dedicated eBusiness team who can initiate and manage the project at each stage of the integration.

With over 150 live B2B projects and over 400 Supply Centres installed in Europe, Life Technologiestm has the experience to meet your integration needs.

A premium service with premium support Our commitment to providing you with 24/7 access to the products you need does not end with on-site stocking. From installation to invoicing, your dedicated Supply Centre team is available to assist with any query you may have.

Why wait? To find out more about how to install a Supply Centre at your site, please contact your local Life Technologies sales representative or contact the Supply Centre team at: [email protected]


216


Compound Formula

Formula weight (to nearest

whole number)

Actinomycin D•5H2O C62H86N12O16•5H2O 1,255

Adenine sulfate•2H2O (C5H5N5)2•H2SO4•2H2O 404

Adenosine C10H13N5O4 267

Adenosine 5´-phosphate C10H14N5O7P 347

Adenosine 5´-triphosphate disodium salt•3H2O

C10H14N5Na2O13P3•3H2O 605

Adenylic acid C10H14N5O7P 347

L-Alanine C3H7NO2 89

L-α-Amino-n-butyric acid C4H9NO2 103

Ammonium nitrate NH4NO3 80

Ammonium molybdate•4H2O (NH4)6Mo7O24•4H2O 1,236

L-Arginine C6H14N4O2 174

L-Arginine hydrochloride C6H14N4O2•HCl 211

Ascorbic acid C6H8O6 176

L-Asparagine C4H8N2O3 132

L-Asparagine•H2O C4H8N2O3•H2O 150

L-Aspartic acid C4H7NO4 133

8-Azaguanine C4H4N6O 152

Biotin C10H16N2O3S 244

Calciferol (Vitamin D2) C28H44O 397

Calcium chloride CaCl2 111

Calcium chloride•2H2O CaCl2•2H2O 147

Calcium lactate C6H10CaO6 218

Calcium nitrate•4H2O Ca(NO3)2•4H2O 236

D-Calcium pantothenate C18H32CaN2O10 477

Cholesterol C27H46O 387

Choline bitartrate C9H19NO7 253

Choline chloride C5H14ClNO 140

Co-carboxylase C12H19ClN4O7P2S 461

Cobalt chloride•6H2O CoCl2•6H2O 238

Coenzyme A C21H36N7O16P3S 768

Colcemid™ * C21H25NO5 371

Cupric chloride•2H2O CuCl2•2H2O 170

Cupric sulfate•5H2O CuSO4•5H2O 250

L-Cysteine C3H7NO2S 121

L-Cysteine hydrochloride•H2O C3H7NO2S•HCl•H2O 176

L-Cystine C6H12N2O4S2 240

L-Cystine dihydrochloride C6H12N2O4S2•2HCl 313

Cytidine C9H13N3O5 243

2´-Deoxyadenosine C10H13N5O3 251

2´-Deoxycytidine C9H13N3O4 227

2´-Deoxycytidine hydrochloride C9H13N3O4•HCl 264

2´-Deoxyguanosine C10H13N5O4 267

Deoxyribose C10H5O4 134

Compound Formula


whole number)

Ergocalciferol (Vitamin D2) C28H44O 397

Ferric nitrate•9H2O Fe(NO3)3•9H2O 404

Ferrous sulfate•7H2O FeSO4•7H2O 278

Flavine C27H33N9O15P2 786

FAD (flavin adenine dinucleotide)

C27H33N9O15P2 786

Folic acid C19H19N7O6 441

Folinic acid, calcium salt C20H21CaN7O7 512

D-Fructose C6H12O6 180

Fumaric acid C4H4O4 116

D(+) Galactose C6H12O6 180

D-Glucosamine hydrochloride C6H13NO5•HCl 216

D-Glucose C6H12O6 180

D-Glucuronolactone C6H8O6 176

GlutaMAX™-I C8H15N3O4 217

GlutaMAX™-II C7H13N3O4 203

L-Glutamic acid C5H9NO4 147

L-Glutamine C5H10N2O3 146

Glutathione sodium salt C10H16N3O6S•Na 329

Glutathione (reduced) C10H17N3O6S 307

Glycine C2H5NO2 75

Guanine hydrochloride C5H5N5O•HCl 188

Guanosine C10H13N5O5 283

HEPES C8H18N2O4S 238

L-Histidine C6H9N3O2 155

L-Histidine hydrochloride•H2O C6H9N3O2•HCl•H2O 210

L-Hydroxyproline C5H9NO3 131

Hypoxanthine C5H4N4O 136

Hypoxanthine sodium salt C5H4N4O•Na 159

i-Inositol C6H12O6 180

L-Isoleucine C6H13NO2 131

Kanamycin sulfate C18H36N4O11•H2SO4 583

α-Ketoglutaric acid C5H6O5 146

L-Leucine C6H13NO2 131

Linoleic acid C18H32O2 280

Lipoic acid C8H14O2S2 206

L-Lysine C6H14N2O2 146

L-Lysine hydrochloride C6H14N2O2•HCl 183

Magnesium sulfate MgSO4 120

Magnesium sulfate•7H2O MgSO4•7H2O 246

Magnesium chloride MgCl2 95

Magnesium chloride•6H2O MgCl2•6H2O 203

Malic acid C4H6O5 134

Maltose C12H12O11 342

* Colcemid™ is a trademark of Ciba Limited.

Formulas and Formula Weights for Commonly Used Medium Components




Compound Formula


whole number)

Manganese chloride•4H2O MnCl2•4H2O 198

Manganese sulfate•H2O MnSO4•H2O 169

Menadione (Vitamin K3) C11H8O2 172

Menadione sodium bisulfate C11H9NaO5S 276

L-Methionine C5H11NO2S 149

5-Methylcytosine C5H7N3O 125

5-Methyl-deoxycytidine C10H15N3O3 225

Methyl linoleate C19H34O2 295

NAD (nicotinamide adenine dinucleotide)

C21H27N7O14P2 663

NADP (nicotinamide adenine dinucleotide phosphate)

C21H28N7O17P3 743

Niacinamide C6H6N2O 122

Nicotinic acid C6H5NO2 123

L-Ornithine hydrochloride C5H13ClN2O2 169

Para-aminobenzoic acid C7H7NO2 137

Phenol red C19H12O5S•Na2 398

Potassium chloride KCl 75

Potassium nitrate KNO3 101

Potassium phosphate monobasic

KH2PO4 136

L-Phenylalanine C9H11NO2 165

L-Proline C5H9NO2 115

Putrescine dihydrochloride C4H14Cl2N2 161

Pyridoxal hydrochloride C8H9NO3•HCl 204

Pyridoxal 5´-phosphate C8H10NO6P 247

Pyridoxine hydrochloride C8H11NO3•HCl 206

Riboflavin C17H20N4O6 376

Riboflavin 5´-phosphate sodium salt

C17H20N4NaO9P 478

D-Ribose C5H10O5 150

L-Serine C3H7NO3 105

Sodium acetate C2H3O2Na 82

Sodium acetate•3H2O C2H3O2Na•3H2O 136

Sodium bicarbonate NaHCO3 84

Sodium chloride NaCl 58

Sodium glucuronate•H2O C6H11NaO7•H2O 236

Compound Formula


whole number)

Sodium phosphate dibasic Na2HPO4 142

Sodium phosphate dibasic•7H2O

Na2HPO4•7H2O 268

Sodium phosphate monobasic•H2O

NaH2PO4•H2O 138

Sodium pyruvate C3H3O3•Na 110

Sodium selenite Na2SeO3 173

Sodium succinate•6H2O C4H4O4Na2•6H2O 270

Succinic acid C4H6O4 118

Sucrose C12H22O11 342

L-Taurine C2H7NO3S 125

Thiamine hydrochloride C12H17ClN4OS•HCl 337

Thiamine monophosphate C12H20N4O8P2S 442

L-Threonine C4H9NO3 119

Thymidine C10H14N2O5 242

Thymine (5-Methyluracil) C5H6N2O2 126

DL-α-Tocopherol acetate C31H52O3 473

DL-α-Tocopherol phosphate sodium salt

C29H55O9P2Na4•2H2O 738

Trehalose•2H2O C12H22O11•2H2O 378

L-Tryptophan C11H12N2O2 204

L-Tyrosine C9H11NO3 181

L-Tyrosine disodium salt C9H9NO3•2Na 225

Uracil C4H4N2O2 112

Uridine C9H12N2O6 244

Uridine 5´-triphosphate C9H15N2O15P3 484

Uridine 5´-triphosphate trisodium salt•2H2O

C9H12N2O15P3Na3•2H2O 586

L-Valine C5H11NO2 117

Vitamin A C20H30O 286

Vitamin A (acetate) C22H32O2 328

Vitamin B12 C63H88CoN14O14P 1,355

Xanthine C5H4N4O2 152

Xanthine sodium salt C5H3NaN4O2 174

Zinc chloride ZnCl2 136

Zinc sulfate•7H2O ZnSO4•7H2O 288


218


Troubleshooting: Cell Culture*Problem Possible cause Suggested solution

Rapid pH shift in medium

Incorrect carbon dioxide (CO2) tension

Overly tight caps on tissue culture flasks Insufficient bicarbonate bufferingIncorrect salts in medium

Bacterial, yeast, or fungal contamination

Increase or decrease percentage of CO2 in the incubator based on concentration of sodium bicarbonate in medium. For sodium bicarbonate concentrations of 2.0 to 3.7 g/L, use CO2 amounts of 5% to 10%, respectively. Switch to CO2-Independent Medium (Cat. no. 18045-088). Loosen caps one-quarter turn. Add HEPES buffer to a final concentration of 10 to 25 mM. Use an Earle’s salts–based medium in a CO2, environment and a Hanks’ salts–based medium in atmo-spheric conditions.Discard culture and medium. Try to decontaminate culture (see Decontami nating Cultures with Antibiotics and Antimycotics, page 207).

Precipitate in medium, no change in pH

Residual phosphate left over from detergent washing, which can precipitate powdered medium components

Frozen medium

Rinse glassware in deionized, distilled water several times, then sterilize.

Warm medium to 37°C and swirl to dissolve. If precipitate remains, discard medium.

Precipitate in medium, change in pH

Bacterial or fungal contamination Discard medium.Try to decontaminate culture (see Decontaminating Cultures with Antibiotics and Antimycotics, page 207).

Cells not adhering to culture vessel

Overly trypsinized cellsMycoplasma contamination

No attachment factors in medium

Trypsinize for a shorter time, or use less trypsin (see Dissociation of Cells from Culture Vessels, page 205). Segregate culture and test for mycoplasma infection. Clean hood and incubator. If culture is contaminated, discard.For serum-free formulations, be sure they contain attachment factors or use coated plates.

Suspension cells clumping together

Presence of calcium and magnesium ions

Mycoplasma contamination

Cell lysis and release of DNA resulting from overdigestion with proteolytic enzymes

Wash cells in a balanced salt solution without calcium and magnesium. Gently pipet cells to obtain a single-cell suspension.Segregate culture and test for mycoplasma infection. Clean hood and incubator. If culture is contaminated, discard.Treat cells with 0.001% DNase I.

Rinse cells with PBS and seed fresh medium.

Primary cell culture is contaminated

Contamination of primary tissue carried over to culture

Wash tissue pieces several times in a balanced salt solution containing a higher concentration of antibiotics and antimycotics before culturing.

Decreased growth of culture

Change in medium or serum

Depletion, absence, or breakdown of essential growth-promoting components such as L-glutamine or growth factors

Low-level bacterial or fungal contaminationImproper storage of reagents

Low initial cell inoculumSenescence of finite cultureMycoplasma contamination

Compare medium formulations for differences in glucose, amino acids, and other components.Compare the old lot of serum with the new lot in a growth experiment. Increase initial cell inoculum. Adapt cells sequentially to the new medium. Remove medium and add fresh medium.

Supplement medium with growth-promoting components.Substitute GlutaMAX™-I (Cat. no. 35050-061) for L-glutamine in the medium. Grow culture without antibiotics. If contaminated, discard culture.Try to decontaminate, (see Decontaminating Cultures with Antibiotics and Antimycotics, page 207).Store sera at –5°C to –20°C. Store media at 2°C to 8°C in the dark. Store complete media at 2°C to 8°C in the dark and use within 2 weeks. Minimize exposure of sera and media to light. Increase viable cell inoculum. Discard culture; obtain new cell stock.Segregate culture and test for mycoplasma infection. Clean hood and incubator. If culture is contaminated, discard.

Death of culture No CO2 in the incubator

Temperature fluctuations in the incubatorUse of Fungizone™ Reagent or antibiotics at toxic concentrations Cell damage during thawing or cryopreservationIncorrect osmotic pressure in medium

Buildup of toxic metabolites in the medium

Monitor rate of CO2 use in incubators to determine when to change tanks. Check line connections fre-quently for leaks. Avoid opening and closing incubator doors.Monitor temperature of incubator.Use less Fungizone™ Reagent (see Use of Antibiotics and Antimycotics, page 207 for appropriate concentrations).† Note: For serum-free media reduce antibiotics 10 times.Obtain another aliquot of cells (see Cryopreservation of Mammalian Cells, page 203).

Check osmolality of complete medium. Note: Most mammalian cells can tolerate an osmolality of 260 to 350 mOsm/kg. Addition of reagents such as HEPES and drugs may affect osmolality. Note: Insect cells require media with a higher osmolality (340 to 380 mOsm/kg) than mammalian cells.Remove medium and add fresh medium.

* There are several common problems encountered when culturing cells. Problems in primary cultures may have different causes than the same problem in established cell lines. Below are some of the common problems encountered when culturing cells, along with their possible causes and suggested solutions. † Fungizone™ is a trademark of E.R. Squibb & Sons, LLC.




Useful Numbers for Cell Culture*

Surface area (mm2) Seeding density Cells at confluency †Versene

(ml of 0.53 mM EDTA)Trypsin (ml of 0.05%

trypsin, 0.53 mM EDTA) Growth medium (ml)

Dishes

35 mm 962 0.3 × 106 1.2 × 106 1 1 2

60 mm 2,827 0.8 × 106 3.2 × 106 3 2 3

100 mm 7,854 2.2 × 106 8.8 × 106 5 3 10

150 mm 17,671 5.0 × 106 20.0 × 106 10 8 20

Cluster plates

6-well 962 0.3 × 106 1.2 × 106 2 2 3–5

12-well 401 0.1 × 106 0.4 × 106 1 1 1–2

24-well 200 0.05 × 106 0.2 × 106 0.5 0.5 0.5–1.0

Flasks

T-25 2,500 0.7 × 106 2.8 × 106 3 3 3–5

T-75 7,500 2.1 × 106 8.4 × 106 5 5 8–15

T-160 16,000 4.6 × 106 18.4 × 106 10 10 15–30

* There are various sizes of dishes and flasks used for cell culture. Some useful numbers such as surface area and volumes of dissociation solutions are given below for various sizes of culture vessels. † The number of cells on a confluent plate, dish, or flask will vary with cell type. For this table, HeLa cells were used.

Buffer Systems for Cell CultureBuffered salt solutions for cell culture can be divided into two principal types: those designed to equilibrate with atmospheric conditions, and those designed to equilibrate with a gas phase containing 5% to 10% CO2. Media utilizing Hanks’ salts are designed for atmospheric equilibration, based upon the physiologically relevant pKa of phosphoric acid. These media contain bicarbonate ions for carboxy lation and similar biological processes.1 Use of Hanks’ salts in a CO2 incubator will result in a rapid drop in pH of the culture medium. This will cause the phenol red indicator to turn yellow.

Media based on Earle’s salts are buffered with a bicarbonate/carbonic acid system and rely upon the physiologically relevant pKa for carbonic acid and the equilibration of gaseous and liquid carbon dioxide to maintain the pH in a CO2-equilibrated incubator.1 Use of Earle’s salts in atmospheric conditions will result in a rapid rise in pH of the culture medium. This will cause the phenol red indicator to turn purple.

Reference1. Jayme, D.W. and Blackman, K.E. (1985) Adv. in Biotechnol. Processes 5, 9.

Specialty Media and Applications* Cell type Specialty media Application

A wide range of cells routinely cultured in serum-supplemented medium

Advanced DMEM and Advanced MEM Enhanced standard basal formulations which allow for a 90% reduction in the required serum supplementation (no cell adaptation needed)

Opti-MEM® I Reduced-Serum Medium A modified Eagles’ MEM medium intended to reduce the amount of serum supplementa-tion by ≥50%

Hybridomas with ethanolamine hydrochloride Permits some hybridomas to grow serum-free

Amniocytes, chorionic villus cells AmnioMAX™-II Complete Medium In vitro propagation of primary human amniotic fluid cells for karyotyping

AmnioMAX™-C100 Complete Medium (basal medium and supplement)

In vitro propagation of primary human amniotic fluid cells for karyotyping. Supplement contains growth supplements (serum) and antibiotics

Lymphocytes PB-MAX™ Karyotyping Medium Fully supplemented (including FBS) RPMI-based medium for short-term cultivation of peripheral blood lymphocytes

Bone marrow and peripheral blood MarrowMAX™ Bone Marrow Medium A fully supplemented medium developed specifically to support bone marrow and peripheral blood cell culture for in vitro cytogenetic analysis of hematological disease

* Specialty media are media supplied with or supplemented with serum designed for specific applications.


220


Serum-free Media and ApplicationsSerum-free media are designed to grow a specific cell type or perform a specific application in the absence of serum.

Serum-free media (SFM) represent an important tool that allows cell culture to be done with a defined set of conditions as free as possible of confounding variables.

Advantages of using serum-free media:Increased definition ■

More consistent performance ■

Easier purification and downstream processing ■

Precise evaluations of cellular function ■

Increased growth and/or productivity ■

Better control over physiological responsiveness ■

Enhanced detection of cellular mediators ■

Certain applications may require the addition of growth factors and/or cytokines. Note: Customized cell culture media are available. Submit your requests to our Custom Products Services group for timely evaluation and production of the formulation and delivery system that best fits your needs.

GlossarySerum-free media — gibco® Serum-free Media do not require supple mentation with serum, but may contain discrete proteins or bulk protein fractions.Protein-free media—gibco® Protein-free Media contain no proteins, but may contain plant or yeast hydrolysates. Many are animal origin–free.Chemically defined media—gibco® Chemically Defined Media contain no proteins, hydrolysates, or components of unknown composition. These media are animal origin–free and all components have a known chemical structure.Animal origin–free products—gibco® animal origin–free products do not contain material directly derived from animal tissues, cells, or body fluids of higher eukaryotic organisms, such as mammals (including humans), fish, birds, insects, etc. The term “animal origin” does not pertain to lower eukaryotic organisms such as the higher plants, fungi, protozoa, and algae, nor does it include prokaryotic organisms such as bacteria or blue-green algae.

Hybridoma CultureProduct Optimized for Application Quantity Cat. no.


FREE CD Hybridoma Medium*Human, mouse, rat hybridomas, myelomas, NS0, NS-1, and other steroid-dependent cells when used with 250X Cholesterol Lipid Concentrate

Growth and MAb production

Can be used to express other proteins in engineered myeloma cell lines

1,000 ml

11279-023


FREE CD Hybridoma AGT™ Dry granular format of CD Hybridoma Medium

1 × 1 L 1 × 10 L

12372-025 12372-017

Hybridoma-SFM Low-protein 20 µg/ml

Human, mouse, rat hybridomas, myelomas Growth and MAb production


500 ml 1,000 ml

12045-084 12045-076


FREE PFHM-II Protein-free Hybridoma Medium

Human, mouse, rat hybridomas, myelomas Growth and MAb production


1,000 ml 12040-077

* Drug Master File available.




Chinese Hamster Ovary (CHO) Cell CultureProduct Optimized for Application Quantity Cat. no.


FREE CD OptiCHO™ MediumSuspension CHO cells (including CHO-S cells)

Growth and production of recombinant proteins

1,000 ml 6 × 1,000 ml

12681-011 12681-029


FREE CD CHO Medium* Suspension CHO cells (including CHO-S cells)

Growth and production of recombinant proteins

500 ml 1,000 ml

10743-011 10743-029


FREE CD CHO Medium AGT™ *

Dry granular format of CD CHO Medium

1 × 1 L 1 × 10 L

12490-017 12490-025


FREE CD CHO-A Medium Adherent CHO cells 500 ml 097-0182DJ

CHO-S-SFM II*

Available without hypoxanthine and thymidine (Cat. no. 31033-020)

Suspension CHO cells (including CHO-S cells)

Growth and production of recombinant proteins in suspension culture

500 ml 1,000 ml

12052-114 12052-098


FREE CHO III PFM* Suspension CHO cells (including CHO-S cells)

Growth and production of recombinant proteins in suspension culture

500 ml 096-0334DJ


FREE CHO III-A-PFM Adherent CHO cells Growth and production of recombinant proteins in adherent culture

1,000 ml 097-0147DK

CHO-S Cells, adapted to CD CHO Medium, CHO-S-SFM II and CHO III PFM 1.5 ml 11619-012


Human Embryonic Kidney (293) Cell CultureProduct Optimized for Application Quantity Cat. no.


FREE CD 293 Medium* Suspension 293 cells (including 293-F, 293-H)

Growth and recombinant protein or adenovirus production in suspension culture

1,000 ml 11913-019


FREE CD 293 AGT™ *

Dry granular format of CD 293 Medium

1 × 1 L 1 × 10 L

12529-020 12529-012


FREE 293 SFM II*

Low-protein <10 µg/ml

Suspension 293 cells and HeLa S3 cells

Growth and recombinant protein or adenovirus production in suspension culture

1,000 ml 11686-029


FREE FreeStyle™ 293 Expression Medium Suspension 293-F cells Growth and transfection in suspension culture 1,000 ml 6 × 1,000 ml

8 L fill in 20 L Wave Cellbag®

12338-018 12338-026

0070049BB

293-F Cells, adapted to CD 293 Medium and 293 SFM II 1.5 ml 11625-019

293-H Cells, adapted to CD 293 Medium and 293 SFM II 1.5 ml 11631-017

FreeStyle™ 293-F Cells, adapted to FreeStyle™ 293 Expression Medium 1 × 107 cells R790-07


Mammalian Cell Culture for Virus ProductionProduct Optimized for Application Quantity Cat. no.


FREE VP-SFM* Low-protein <10 µg/ml

Suspension BHK-21 cells, adherent VERO, COS-7L, MDCK, HEp-2

For culture of kidney epithelial and related cells used in virus production

1,000 ml 11681-020


FREE VP-SFM AGT™ * Dry granular format of VP-SFM

1 × 1 L 1 × 10 L

12559-027 12559-019


FREE OptiPRO™ SFM* Low protein <10 µg/ml

Adherent MDCK, VERO, PK-15, MDBK, BHK-21, and related cells used in virus production

For culture of kidney epithelial and related cells used in virus production

1,000 ml 12309-019


FREE Protein Expression Medium (PEM)

Recombinant proteins in PER.C6® cells † Supports growth of parental PER.C6®, HEK293, and HeLa cells in suspension culture

1,000 ml 12661-013

* Drug Master File available. † PER.C6® is a trademark of Crucell Holland B.V.


222


Insect Cell CultureProduct Optimized for Application Quantity Cat. no.

Express Five® SFM Suspension BTI-TN-5B1-4 insect cells Growth and maintenance of cells used for the baculovirus expression vector system (BEVS) for adherent or suspension culture

Large-scale production of recombinant protein expressed by BEVS

1,000 ml 10486-025

Sf-900™ II SFM* Suspension Sf9, Sf21 (Spodoptera frugiperda), TN368 cells (Trichoplusia ni)

Growth and maintenance of cells used for BEVS for adherent or suspension culture

Large-scale production of recombinant protein expressed by BEVS

500 ml 1,000 ml

6 × 1,000 ml (case) 5 L

10 L 20 L

10 L fill in 20 L Wave Cellbag®

10902-096 10902-088 10902-104 10902-161 10902-070 10902-187 0070037BA

D.Mel-2 Cells 1.5 ml 10831-014

High Five™ Cells, adapted to Express Five® SFM 3 × 106 cells/ml B855-02

Sf9 Cells, adapted to Sf-900™ II SFM 1.5 ml 11496-015

Sf21 Cells, adapted to Sf-900™ II SFM 1.5 ml 11497-013


Neuronal Cell CultureProduct Optimized for Application Quantity Cat. no.

Neurobasal™ Medium Fetal neurons Basal medium lacking excitatory amino acids used in conjunction with supplements to make a complete serum-free medium

Long-term growth of neurons

500 ml 21103-049

Neurobasal™ Medium without phenol red

500 ml 12348-017

Neurobasal™-A MediumAdult and postnatal neurons (>1 week old)

500 ml 10888-022

Neurobasal™-A Medium without phenol red

500 ml 12349-015

B-27 Serum-free Supplement

Primary embryonic hippocampal neu-rons; primary neurons from striatum, substantia nigra, septum

Growth and maintenance. Minimizes glial cell proliferation

B-27 minus AO to study free-radical damage, apoptosis (B-27 minus AO is formulated without any cortical antioxidants)

10 ml 17504-044

B-27 Supplement minus AO 10 ml 10889-038

B-27 Supplement minus Vitamin A (Retinoic acid)

Study growth of CNS progenitor or stem cells 10 ml 12587-010

N-2 Supplement Primary embryonic hippocampal neu-rons, tumor cell lines of neuronal origin (PC12, B104, N1E-115 and NS20)

Maintenance of primary neurons (low protein, <125 µg/ml)

Growth and maintenance of neuronal tumor cell lines

5 ml 17502-048

G-5 Supplement Primary glial cells, tumor cell lines of glial origin (U-251, MGsp, C62BD, RN-22), astrocytes, microglia, oligodendrocytes

Growth and maintenance of primary and serial tumor glial cells 1 ml 17503-012




Blood and Bone Marrow CultureProduct Optimized for Application Quantity Cat. no.

AIM V® Medium, liquid,* research grade, with HSA

Lymphocytes, macrophages, monocytes, lymphoid cell lines

Research use only, growth of TIL cells, CTL cells, and monocytes 500 ml 1,000 ml

12055-091 12055-083

AIM V® Medium, liquid research grade, with BSA

Lymphocytes, macrophages, monocytes, and lymphoid cell lines

Research use only, growth of TIL cells, CTL cells, and monocytes 500 ml 31035-025

AIM V® Medium, liquid, therapeutic grade

Growth of tumor infiltrating lymphocytes (TIL cells), cytotoxic T cells, monocytes, and ex vivo activation

1,000 ml 10 L

087-0112DK 087-0112BK

Macrophage-SFM Macrophages, monocytes Growth and maintenance (addition of GM-CSF may be necessary). Demon stration of macrophage phagocytosis. Activation of cells to kill tumor cells with γ-interferon or lipopolysaccharide supplementation

500 ml 12065-074

StemPro®-34 SFM Supplied with Hematopoietic StemPro® Nutrient Supplement

Human hematopoietic progenitor cells (CD34+) from bone marrow, peripheral blood, or neonatal cord blood

Growth and maintenance of human hematopoietic progenitor cells (addition of factors necessary). Study synergistic/individual effects of growth

500 ml 10639-011

MarrowMAX™ Bone Marrow Medium

Bone marrow, peripheral blood, and hematopoietic cells

Growth of bone marrow, peripheral blood, and hematopoietic cells in short-term in vitro culture

100 ml 12260-014

* Drug Master File available

Other Mammalian Cell CultureProduct Optimized for Application Quantity Cat. no.

Human Endothelial-SFM* Primary and secondary human umbili-cal venous, microvascular, and arterial endothelial cells

Growth and maintenance to study cell-cell interactions, injury analysis, atherosclerosis, signal transduction, cytokine production, and cell matrix interaction. Requires supplementation with bFGF, EGF, and fibronectin (sold separately)

500 ml 11111-044

Hepatozyme-SFM Primary human, monkey, and rat hepatocytes

Maintenance of hepatocytes (cytochrome P450 induction main-tained >9 days)

500 ml 17705-021

Keratinocyte-SFM (with EGF, BPE)* Human epidermal keratinocytes and cervical epithelial cells (will not support fibroblast or melanocyte cells)

Growth and maintenance for dermal substitutes, gene therapy, and in vitro toxicology

500 ml 17005-042

Keratinocyte-SFM (without CaCl2)* Without bovine pituitary 500 ml 37010-022

Defined Keratinocyte-SFM* Human epidermal keratinocytes cervi-cal epithelial cells (will not support fibroblast or melanocyte cells)

Low-protein (<25 µg/ml) without BPE medium for cultivation of keratinocytes. Can be used for cervical epithelial cells for studies involving the human papilloma virus

500 ml 10744-019

Primary Human Keratinocytes, adapted to Defined Keratinocyte-SFM 1 ml 12332-011


Stem Cell CultureProduct Optimized for Application Quantity Cat. no.

KnocKout™ DMEM Murine and human embryonic stem (ES) cells

Growth and maintenance of undifferentiated ES cells for production of transgenic mice. Growth and maintenance of both human and murine ES cells used in differentiation studies

500 ml 10829-018

KnocKout™ Serum Replacement 500 ml 10828-028

StemPro® hESC SFM Growth and expansion of human embryonic stem cells (hESCs)

Maintains pluripotency in a growing list of hESC lines including BG01, BG02, BG03, H1, H9, and BG01V

1 kit A1000701

MesenPRO RS™ Medium Growth of human mesenchymal stem cells (MSCs) in culture

Improvement of MSC expansion. Expansion of cells for multiple pas-sages while maintaining multipotential phenotype (i.e., differentiation into osteogenic, chondrogenic, and adipogenic lineages)

1 kit 12746-012


224


Adaptation of Cell Cultures to a Serum-free MediumThere are two approaches to adapt cells to serum-free medium (SFM):1. Direct adaptation—cells switched from serum-supplemented medium

into SFM.2. Sequential adaptation or weaning—cells switched from serum-

supplemented medium into SFM in several steps. (Sequential adaptation tends to be less harsh on cells than direct adaptation.)

To adapt cells to SFM, it is critical that the culture is:In mid-logarithmic phase ■

>90% viable ■

Seeded at a higher initial cell inoculum during adaptation ■

Below are general protocols for adaptation. Initial cell inocula, cell yields, and culture periods are intended as starting points for subsequent optimi zation.

Direct AdaptationSome cells can be directly adapted from serum-containing medium to SFM. For direct adaptation, the cell inoculum should be 2.5 × 105 to 3.5 × 105 cells/ml. Subculture cells when the cell density is 1 × 106 to 3 × 106 cells/ml. Cells are fully adapted to SFM when the cell density is 2 × 106 to 4 × 106 cells/ml after 4 to 7 days in culture. Stock cultures of cells adapted to SFM should be subcultured in SFM every 3 to 5 days when the cell density is 1 × 106 to 3 × 106 with 90% viability.

Sequential AdaptationSequential adaptation is Invitrogen’s preferred method for adapting cells to serum-free media (SFM), with a typical conversion being:

Passage 1 75% serum-supplemented medium: 25% SFM



Passage 4 100% SFM

Because the change from 75% to 100% SFM may be too stressful for your cells, you may need to carry the cells for 2–3 passages in a 10% serum-supplemented medium: 90% SFM mixture. Most cell lines can be considered fully adapted after 3 passages in 100% SFM. Occasionally you may have trouble getting your cells past a certain step even before going 100% SFM. If this happens, go back and passage the cells 2–3 times in the previous ratio of serum-supplemented medium to serum-free medium.

Points to Consider in Serum-free CultureOverall, cells in serum-free culture are more sensitive to extremes of pH, temperature, osmolality, mechanical forces, and enzyme treatment.

AntibioticsIt is best not to use antibiotics in serum-free media. If you do, we recommend that you use 5- to 10-fold less than you would in a serum-supplemented medium. This is because serum proteins tend to bind a certain amount of the antibiotic added; without these serum proteins the level of antibiotic may be toxic to certain cells.

Higher DensityCells must be in the mid-logarithmic phase of growth with viability >90% prior to adaptation. Sequential adaptation may be necessary. Seeding cultures at a higher density than normal at each passage during SFM adaptation may help the process. Because some percentage of cells may not survive in the new culture environment, having more cells present will increase the number of viable cells to further passage.

ClumpingCell clumping often occurs during adaptation to SFM. We recommend that you gently triturate the clumps to break them up when passaging cells.

MorphologyIt is not uncommon to see slight changes in cellular morphology during and after adaptation to SFM. As long as doubling times and viability remain good, slight changes in morphology should not be a reason for concern.

Adaptation with Conditioned MediumAn alternate method for adaptation to SFM involves the use of “condi-tioned medium.” This is medium the cells have been growing in for one full passage. If you choose this method, you can facilitate adaptation as follows:

Passage 1 100% serum-supplemented medium

Passage 2 50% medium from passage 1: 50% SFM



Passage 5 100% SFM

Whichever adaptation method you choose, we strongly recommend that you always take these precautions:

Make a frozen stock of the cells in the serum-supplemented medium ■

prior to adaptation.Keep a culture going of the cells in each prior condition when starting ■

the next level of adaptation as a fallback if the cells do not survive in the next passage.




Adaptation of Cell Cultures to an Alternative Serum

Adaptation of Cells from Monolayer to Suspension CultureAdaptation of adherent cells to suspension culture may be required to grow cells to a higher density or for the growth of cells in serum-free medium. This protocol describes the procedure for cells grown in T-25 or T-75 flasks. Approximately 6 to 8 T-75 flasks provide enough HeLa cells (5 × 107) to seed a suspension culture in a spinner or shake flask. Note: The number of starting flasks will vary depending on the cell line. For best results remember the following:

Use cells 50% to 80% confluent ■

Use cells with >90% viability ■

1. Remove growth medium.2. Wash cells using 1X D-PBS without calcium and magnesium (3 ml for

T-25 flask or 5 ml for T-75 flask). Discard the wash solution.3. Add trypsin-EDTA (0.05% trypsin, 0.53 mM EDTA•4Na; 3 ml for T-25

flask or 5 ml for T-75 flask) to the side of the flask opposite the cells. Briefly swirl the flask. Discard all but enough trypsin-EDTA to leave a thin film of liquid over the cells. Incubate the cells at 37°C for 5 to 10 min. Monitor the cells during this incubation under a microscope. When they round up, tap the flask to dislodge the cells.

4. Add growth medium (6 ml for T-25 flask or 10 ml for a T-75 flask) to the cell suspension.

Note: For serum-free medium, add soybean trypsin inhibitor. Usu-ally, using 1:1 (v:v) 0.25 mg/ml trypsin inhibitor solution to trypsin will inhibit trypsin.

5. Transfer the cell suspension to a 15 ml centrifuge tube. Centrifuge at 100 × g for 4 min. Remove the supernatant (trypsin-EDTA mixed with growth medium) by aspiration.

6. Resuspend the cells in 5 ml of growth medium. Centrifuge at 100 × g for 4 min. Remove the supernatant by aspiration.Note: For serum-free medium resuspend the cells in D-PBS without calcium or magnesium.

7. Resuspend the cells in 5 ml of growth medium. Count the number of viable cells.

8. Dilute cells in growth medium to obtain a density of 5 × 105 cells/ml.9. Subculture in a sterile spinner or shake flask. Leave sufficient

headspace for adequate gas exchange (100 ml of medium in a 250 ml spinner flask or 75 to 100 ml of medium in a 250 ml shake flask).

Note: For serum-free medium that does not contain a shear protectant, add Pluronic® F-68 to a final concentration of 0.1%.

10. Loosen caps for gas exchange and place at 37°C in a humidified atmosphere of 5% to 10% CO2 in air. For spinner flasks, use an impeller speed of 75 to 95 rpm. For shake flasks, rotate on a shaker platform at 125 to 135 rpm.

11. Check viable cell density daily to establish growth kinetics in suspen-sion culture.

12. When the cell density is 1 × 106 cells/ml (or on day 4 post-planting) passage the cells at a density of 5 × 105 cells/ml.

Note: Centrifugation and the addition of fresh growth medium may be necessary if the cell density has not reached 1 × 106 cells/ml by day 4.

13. Once cell density reaches 1 × 106 cells/ml with 90% viability by day 3 post-planting for 3 passages, the cells are considered adapted to suspension culture. Seeding density can be reduced to 2 × 105 to 3 × 105 cells/ml.


Many cells adapt best with a sequential method of adaptation. Culture cells with a 1:1 (v:v) blend of the current medium with FBS and the medium with the alternative serum. Reduce the amount of current medium over successive passages by blending the media as follows: 1:2, 1:4, 1:16, and 100% medium with alternative. Passage cells 2 or 3 times with each change in serum composition for adaptation.

Cultures may be switched to the alternative serum directly from FBS. Initially, use the alternative serum at the same concentration as the FBS. A lag in growth may occur. Allow 2 to 3 passages for the growth rate to return to its previous level.


226


Stability of GlutaMAX™-I vs. L-GlutamineGlutamine is an essential nutrient in cell cultures for energy production as well as protein and nucleic acid synthesis. A drawback to glutamine in cell culture media is spontaneous breakdown once in solution. Glutamine breakdown generates ammonia as a by-product, which is toxic to the cells 1 and can affect protein glycosylation.2,3 One strategy to minimize generation of ammonia is to replace the L-glutamine in media with the glutamine dipeptide, GlutaMAX™-I. Figures 1 and 2 demonstrate the improved stability of the glutamine dipeptide and reduction of spontaneous ammonia generation.

An additional benefit to using GlutaMAX™-I in place of L-glutamine in cell culture is the extension of culture time, potentially reducing the number of times the cells must be passaged. Figure 3 is a comparison of MDBK cells cultured in DMEM with 10% fetal bovine serum (FBS) and either glutamine or GlutaMAX™-I. Cells cultured in GlutaMAX™-I reach peak density two days later, and viability declines less rapidly than in cultures with glutamine supplementation.

Many widely used gibco® media formulations are available with the GlutaMAX™ dipeptide substituted for L-glutamine. See page 24 for a complete listing of products. Additionally, the GlutaMAX™ dipeptide is available as a 200 mM solution and can be substituted for glutamine on an equimolar basis. It can be used with most cell lines with equi valent performance and substituted in both serum-supplemented and serum-free formulations requiring L-glutamine.

The slight increase of the lag phase is attributed to the time needed to release the peptidase and digest the dipeptide. This allows a gradual increase in availability of L-glutamine to the cells.1

References1. Hassell, T., Gleave, S., and Butler, M. (1991) Growth Inhibition in Cell Culture. Applied Biochemistry and Biotech-

nology, v. 30, pp 30–41.2. Yang, M., and Butler, M. (2002) Effects on Ammonia and Glucosamine on the Heterogeneity of Erythropoietin

Glycoforms. Biotechnology Progress, v. 18, 129–138.3. Yang, M., and Butler, M. (2000) Effect of Ammonia on the Glycosylation of Human Recombinant Erythropoietin

in Culture. Biotechnology Progress, v. 16, 751–759.

Common Cell Lines Cultured with GlutaMAX™-IMDBK Bovine kidney

MDCK Canine kidney

BHK Hamster kidney

CHO Hamster ovary

C-33 A Human cervical cancer

HT-29 Human colon cancer

293 Human embryonic kidney

Per.C6® * Human embryonic retinoblastoma

BG01v Human ES cell

H1 Human ES cell

HepG2 Human hepatoblastoma used in AlgiMatrix™

HELA Human ovary

AE-1 Mouse hybridoma

3D9 Mouse hybridoma

Ns0/1 Myeloma

Sp2/0 Myeloma

D3 Mouse ES cell lines

J1 Mouse ES cell lines

Jurkat Human T cell leukemia

Swiss 3T3 Swiss mouse fibroblast

* PER.C6® is a trademark of Crucell Holland B.V.

Figure 1—Stability of GlutaMAX™-I vs. L-glutamine in DMEM at 37°C. DMEM was supplemented with GlutaMAX™-I or L-glutamine, aliquoted into vials, and stored at 37°C. Samples were taken daily and frozen at –20°C. Levels of GlutaMAX™-I and L-glutamine were determined by HPLC.

Figure 2—Ammonia levels in supplemented media stored at 37°C. DMEM was supplemented with GlutaMAX™-I or L-glutamine, aliquoted into vials, and stored at 37°C. Samples were taken daily and frozen at –20°C. Levels of ammonia were deter-mined by HPLC.

Figure 3—Growth of MDBK cells in DMEM supple-mented with L-glutamine or GlutaMAX™-I and 10% FBS. MDBK cells were seeded at approximately 1 × 105 cells/flask in DMEM with 10% FBS and L-glu-tamine or GlutaMAX™-I in 25 cm2 T-flasks.

Day0 1 2 3 4 5 6 7

10

9

8

7

6

5

4

3

2

1

0

GlutaMAX™-I

L-glutamine

Tota

l cel

l cou

nt (×

105 )

Am

mon

ia (m

g/L)

Day0 1 2 3 4 5 6 7

70

60

50

40

30

20

10

0

GlutaMAX™-I

L-glutamine

mg/

L

Day

1,000

900

800

700

600

500

400

300

200

0 1 2 3 4 5 6 7

GlutaMAX™-IL-glutamine




Cryopreservation of Mammalian CellsMammalian cells are cryopreserved to avoid loss by contamination, to minimize genetic change in continuous cell lines, and to avoid aging and transformation in finite cell lines. Before cryopreserving, cells should be characterized and checked for contamination.

Recovery™ Cell Culture Freezing Medium (Cat. no. 12648-010) is a complete cryopreservation medium for mammalian cell cultures. With Recovery™ Cell Culture Freezing Medium, you will increase cell viability an average of 25% and save time without measuring and mixing as required to prepare your own cryoprotectant. Recovery™ Cell Culture Freezing Medium is prepared in DMEM (high glucose) with FBS and calf serum and 10% DMSO.

There are several common media used to freeze cells. For serum- con taining medium, the constituents may be as follows:

Complete medium containing 10% glycerol ■

Complete medium containing 10% (dimethylsulfoxide) (DMSO) ■

50% cell-conditioned medium with 50% fresh medium with ■

10% glycerol50% cell-conditioned medium with 50% fresh medium with 10% ■

DMSOFor serum-free medium, some of the common medium constituents may be:

50% cell-conditioned, serum-free medium with 50% fresh serum-free ■

medium containing 7.5% DMSOfresh, serum-free medium containing 7.5% DMSO and 10% cell ■

culture–grade BSA

Suspension Cultures1. Count the number of viable cells to be cryopreserved. Cells should be

in log phase. Centrifuge the cells at ~200 to 400 × g for 5 min to pellet cells. Using a pipette, remove the supernatant down to the smallest volume without disturbing the cells.

2. Resuspend cells in freezing medium at 1 × 107 to 5 × 107 cells/ml for serum-containing medium, or 0.5 × 107 to 1 × 107 cells/ml for serum-free medium.

3. Aliquot into cryogenic storage vials. Place vials on wet ice or in a 4°C refrigerator, and start the freezing procedure within 5 min.

4. Cells are frozen slowly at 1°C/min. This can be done by programmable coolers or by placing vials in an insulated box in a –70°C to –90°C freezer, then transferring to liquid nitrogen storage.1

Adherent Cultures1. Detach cells from the substrate with dissociating agents. Detach as

gently as possible to minimize damage to the cells (see Dissociation of Cells from Culture Vessels, page 204).

2. Resuspend the detached cells in complete growth medium and establish the viable cell count.

3. Centrifuge at ~200 × g for 5 min to pellet cells. Using a pipette, withdraw the supernatant down to the smallest volume without disturbing the cells.

4. Resuspend the cells in freezing medium at 5 × 106 to 1 × 107 cells/ml.

5. Aliquot into cryogenic storage vials. Place vials on wet ice or in a 4°C refrigerator, and start the freezing procedure within 5 min.

6. Cells are frozen slowly at 1°C/min. This can be done by programmable coolers or by placing vials in an insulated box in a –70°C to –90°C freezer, then transferring to liquid nitrogen storage.1

Reference1. Freshney, R. (1987) Culture of Animal Cells: A Manual of Basic Technique, p. 220,

Alan R. Liss, Inc., New York.

Thawing of Cryopreserved CellsCr yopreser ved cel ls are fragi le and require gentle handl ing. Cryopreserved cells are thawed quickly and plated directly into complete growth medium. If cells are particularly sensitive to cryopreservative (DMSO or glycerol), they are centrifuged to remove cryopreservative and then plated into complete growth medium. The following are suggested procedures for thawing cryopreserved cells.

Direct Plating Method1. Remove cells from storage and thaw quickly in a 37°C water bath.2. Plate cells directly with complete growth medium. Use 10 to 20 ml of

complete growth medium per 1 ml of frozen cells. Perform a viable cell count. Cell inoculum should be at least 3 × 105 viable cells/ml.

3. Culture cells for 12 to 24 hr. Replace medium with fresh complete growth medium to remove the cryopreservative.

Centrifugation Method1. Remove cells from storage and thaw quickly in a 37°C water bath.2. Place 1 to 2 ml of frozen cells in ~25 ml of complete growth medium.

Mix very gently.3. Centrifuge the cells at ~80 × g for 2 to 3 min.4. Discard supernatant.5. Gently resuspend the cells in complete growth medium and perform

a viable cell count.6. Plate the cells. Cell inoculum should be at least 3 × 105 viable cells/ml.


228


Dissociation of Cells from Primary TissueA common method to obtain single-cell suspensions from primary tissue is enzymatic disaggregation. Expose the cells to enzymes for a minimal amount of time to preserve maximum viability. The following procedures disaggregate whole tissue to obtain a high yield of viable cells.1

Trypsin1. After dissecting off unusable tissue, mince the remaining tissue into

3 to 4 mm pieces with a sterile scalpel or scissors. Wash the tissue pieces by resuspending in a balanced salt solution without calcium and magnesium. Allow the tissue pieces to settle, and remove the supernatant. Repeat the wash 2 or 3 times.

2. Place the container with the tissue pieces on ice, and remove any remaining supernatant. Add 0.25% trypsin in a balanced salt solution without calcium or magnesium (~1 ml of trypsin for every 100 mg of tissue).

3. Incubate at 4°C for 6 to 18 hr to maximize penetration of the enzyme with little trypsin activity.

4. Decant and discard the trypsin from the tissue pieces. Incubate the tissue pieces with residual trypsin at 37°C for 20 to 30 min.

5. Add warm complete medium to the tissue pieces and gently dis-perse the tissue by pipetting. If using a serum-free medium, also add soybean trypsin inhibitor.

6. Filter the cell suspension through sterile stainless steel mesh (100 to 200 µm) to completely disperse any remaining tissue. Count and seed the cells for culture.

Collagenase1. Mince tissue into 3 to 4 mm pieces with a sterile scalpel or scissors.

Wash the tissue pieces several times with Hanks’ Balanced Salt Solu-tion (HBSS).

2. Add collagenase (50 to 200 units/ml in HBSS).

3. Incubate at 37°C for 4 to 18 hr. Addition of 3 mM CaCl2 increases the efficiency of dissociation.

4. Filter the cell suspension through a sterile stainless steel or nylon mesh to separate the dispersed cells and tissue fragments from the larger pieces. Fresh collagenase can be added to the fragments if further disaggregation is required.

5. Wash the suspension several times by centrifugation in HBSS.6. Resuspend the pellet in culture medium. Count and seed the cells for

culture.

Dispase1. Mince tissue into 3 to 4 mm pieces with a sterile scalpel or scissors.

Wash the tissue pieces several times in a calcium- and magnesium-free balanced salt solution.

2. Add dispase (0.6 to 2.4 units/ml in calcium- and magnesium-free balanced salt solution).

3. Incubate at 37°C for 20 min to several hours.4. Filter the cell suspension through sterile stainless steel or nylon

mesh to separate the dispersed cells and tissue fragments from the larger pieces. Fresh dispase can be added to the fragments if further disaggregation is required.

5. Wash the suspension several times by centrifugation in the balanced salt solution.

6. Resuspend the pellet in culture medium. Count and seed the cells for culture.

Reference1. Freshney, R. (1987) Culture of Animal Cells: A Manual of Basic Technique, p. 117,

Alan R. Liss, Inc., New York.




Dissociation of Cells from Culture VesselsThe following is a general procedure to rapidly remove various cell lines from the substratum while maintaining cellular integrity. This procedure is not meant to be universally applicable for all cell lines. The optimal conditions and concentrations employed for individual systems should be determined empirically.

Monitor cell viability at the time of subculturing ■

Cell viability should be greater than 90% ■

For serum-free media, it is advisable to decrease the amount of trypsin ■

used1. Remove and discard the spent cell culture medium.2. Wash the cells using a balanced salt solution without calcium and

mag nesium or wash with EDTA (see table on next page to determine the correct wash solution). Add the wash solution to the side of the flask opposite the cells. Rinse the cell sheet by rocking the flask for 1 to 2 min and discard wash solution.

3. Add the dissociation solution of choice (Table 1, next page) at 2 to 3 ml/25 cm2 to the side of the flask opposite the cells. Be sure that the dissociation solution covers the cell sheet. Incubate the flask at 37°C. Rock the flask gently. Generally, cells are dissociated in 5 to 15 min. The time needed to dissociate cells will vary according to cell line. Monitor the process carefully to avoid cell damage. Flasks of cell lines that are difficult to remove from the substratum may be tapped to expedite removal.

4. When the cells are completely detached, stand the flask in the upright position to allow the cells to drain to the bottom of the flask. Add com-plete medium to the flask. Disperse the cells by pipetting repeatedly over the surface of the monolayer. Count and subculture the cells. Note: For serum-free media, add soybean trypsin inhibitor. Usually, using 1:1 (v:v) 0.25 mg/ml trypsin inhibitor solution to trypsin will inhibit trypsin.

TrypLE™ ProductsTrypLE™ products are based on a recombinant enzyme that replaces animal trypsin for the dissociation of adherent mammalian cells from plastic. It can be stored refrigerated and is stable at room temperature for six months. TrypLE™ Express works on a wide range of cells and requires little or no protocol changes. Inactivation with trypsin inhibitors is not required.1. Decant the medium from the flask. Rinse the flask with 5 ml of

Dulbecco’s Phosphate Buffered Saline (D-PBS) without calcium or magnesium (base cat. no. 14190). Decant the D-PBS.

2. Add an appropriate volume (i.e., 2 ml in a 75 cm2 flask) of prewarmed TrypLE™ to the flask. Rock the vessel to coat the cell sheet completely.

3. Incubate at 37°C until the cells have detached (observe at 5 minute intervals). Gently rap the vessel to dislodge the cells.

4. Dilute in 2 to 5 ml of cell culture growth medium and transfer the cell suspension to a 15 ml centrifuge tube.

5. Centrifuge for 5 to 10 minutes at 100 × g. Discard the supernatant and suspend the cell pellet in 2 to 5 ml of fresh growth medium.

6. Determine the viable and total counts.7. Seed the flasks according to normal protocol.8. Incubate and subculture according to normal protocol depending on

your cell type.Note: Use of soybean trypsin inhibitor is not recommended.


230


Table 1—Dissociation of cells from culture vessels.

Culture type Wash solution Dissociation solution Products Cat. no.

Most continuous cell lines

Strongly adherent continuous cell lines

Many cells at early passage

HBSS or PBS without calcium and magnesium

D-PBS

Trypsin 0.25% in balanced salt solution without calcium and magnesium

TrypLE™ products

0.25% Trypsin (liquid) 2.5% Trypsin (liquid) (10X) Trypsin Powder (1:250)

TrypLE™ Select (liquid) TrypLE™ Express (liquid)

15050-065 15090-046 27250-018

12563-011 12605-010

Continuous cell lines where integrity of cell surface proteins is important


Trypsin 0.05% in 0.53 mM EDTA

TrypLE™ products

0.05% Trypsin 0.53 mM EDTA (liquid)

0.5% Trypsin 5.3 mM EDTA (10X) (liquid) TrypLE™ Select (liquid) TrypLE™ Express (liquid)

25300-054

15400-054 12563-011 12605-010

Weakly adherent epithelial cells

Transformed fibroblasts

Primary cells where integrity of cell surface proteins is required


EDTA, glycerol in sodium citrate

TrypLE™ products

Enzyme-free Cell Dissociation Buffers Hanks’ based PBS based

TrypLE™ Select (liquid) TrypLE™ Express (liquid)

13150-016 13151-014

12563-011 12605-010

Strongly adherent early-passage cell lines


Trypsin 0.25% in 1 mM EDTA

Dispase 0.6 to 2.4 units/ml in PBS

0.25% Trypsin 1 mM EDTA (liquid) Trypsin Powder (1:250)

Dispase

25200-056 27250-018

17105-041

Epithelial cells* 0.5 mM to 1 mM EDTA 0.5 mM to 1 mM EDTA

Dispase 0.6 to 2.4 units/ml in PBS

Versene 1:5,000 (0.53 mM EDTA in PBS)

Dispase

15040-066

17105-041

Strongly adherent cells*

Epithelial cells

Some tumor cells

0.5 mM to 1 mM EDTA Trypsin 0.25% with 1 mM EDTA

Dispase 0.6 to 2.4 units/ml in PBS without calcium and magnesium

0.25% Trypsin 1 mM EDTA

Versene 1:5,000 (0.53 mM EDTA in PBS)

Trypsin Powder (1:250) Dispase

25200-056

15040-066

27250-018 17105-041

Thick cultures, multiple layers*

Dense cultures that are collagen rich

1 mM EDTA Trypsin 0.25%

Collagenase 200 units/ml, 1 mM EDTA in balanced salt solution without calcium and magnesium

0.25% Trypsin 1 mM EDTA Collagenase 17100, 17101, 17102, 17104

Trypsin Powder (1:250)

25200-056

27250-018

All adherent cultures HBSS or PBS without calcium and magnesium

Scrape off cell sheet †

— —

* Some cells may be sensitive to EDTA. † Scraping may cause mechanical damage and will not yield a single-cell suspension.Note: Adapted from Freshney, R. (1987) Culture of Animal Cells: A Manual of Basic Technique, p. 129, Alan R. Liss, Inc., New York.




Decontaminating Cultures with Antibiotics and AntimycoticsWhen an irreplaceable culture becomes contaminated, researchers may attempt to eliminate or control the contamination. First, determine if the contamination is bacteria, fungus, mycoplasma, or yeast. Isolate the contaminated culture from other cell lines. Clean incubators and laminar flow hoods with a laboratory disinfectant, and check HEPA filters.

Antibiotics and antimycotics at high concentrations can be toxic to some cell lines; therefore, perform a dose-response test to determine the level at which an antibiotic or antimycotic becomes toxic. This is particularly important when using an antimycotic such as Fungizone™ Reagent or an antibiotic such as tylosin. The following is a suggested procedure for determining toxicity levels and decontaminating cultures:1. Dissociate, count, and dilute the cells in antibiotic-free medium. Dilute the cells to the

concentration used for regular cell passage.2. Dispense the cell suspension into a multiwell culture plate or several small flasks. Add the

antibiotic of choice to each well or flask in a range of concentrations. For example, for Fungizone™ Reagent, 0.25, 0.50, 1.0, 2.0, 4.0, and 8.0 µg/ml.

3. Observe the cells daily for signs of toxicity such as sloughing, appearance of vacuoles, decrease in confluency, and rounding.

4. When the toxic antibiotic level has been determined, culture the cells for 2 to 3 passages using the antibiotic at a concentration 1 to 2 times lower than the toxic concentration.

5. Culture the cells for 1 passage in antibiotic-free medium. 6. Repeat Step 4.7. Culture the cells in antibiotic-free medium for 4 to 6 passages to determine if the contamination

has been eliminated.


Use of Antibiotics and Antimycotics*

Antibiotic Recommended concentration Antibiotic spectrumStability in tissue culture

media at 37°C

Fungizone™ Reagent (Amphotericin B)

Gentamicin sulfate

0.25–2.5 µg/ml

5–50 µg/ml

Fungi and yeasts

Gram-positive and gram-negative bacteria and mycoplasmas

3 days

5 days

Kanamycin sulfate

Neomycin sulfate

100 µg/ml

50 µg/ml

Gram-positive and gram-negative bacteria and mycoplasmas

Gram-positive and gram-negative bacteria

5 days

5 days

Penicillin G

Polymixin B sulfate

Streptomycin sulfate

50–100 units/ml

100 units/ml

50–100 µg/ml

Gram-positive bacteria

Gram-negative bacteria

Gram-positive and gram-negative bacteria

3 days

5 days

3 days

The decision to use antibiotics to prevent contamination should be based on the individual researcher’s needs and experience. This table is a general guide for use of gibco® antibiotics in tissue culture media. The concentrations given are for tissue culture media containing serum; serum-free media generally require lower concentrations (1⁄5 to 1⁄10). Also available are solu-tions that use one or more antibiotics in conjunction with an antimycotic. For all medium types, optimal concentrations of anti biotics and antimycotics should be determined empirically.



232


Conversion Factors, Units, and AbbreviationsConversion Factorsbase mass

1 kb ds DNA (Na+) = 6.6 × 105 Da

1 kb ss DNA (Na+) = 3.3 × 105 Da

1 kb ss RNA (Na+) = 3.4 × 105 Da

1 MDa ds DNA (Na+) = 1.52 kb

average mass of dNMP = 330 Da

average mass of dNMP bp = 660 Da

base protein1 kb coding capacity = 333 amino acids = 40,000 Da protein

10,000 Da protein = ~270 bp DNA

50,000 Da protein = ~1.35 kb DNA

100,000 Da protein = ~2.7 kb DNA

average mass of amino acid = 120 Da

mass of nucleic acid moles of nucleic acid1 µg/ml of nucleic acid = 3.0 µM phosphate

1 µg of a 1 kb DNA fragment = 1.5 pmol; 3.0 pmol ends

1 pmol of a 1 kb DNA = 0.66 µg

1 µg pBR322 = 0.36 pmol DNA

1 pmol pBR322 = 2.8 µg

35 nmol of a 24-mer = 277 µg

absorbance of nucleic acid concentration of nucleic acidNucleic acid Concentration (µg/ml) for 1 A260 unit

ds DNA 50

ss DNA 33

ss RNA 40

mass of protein moles of proteinMolecular weight (Da) 1 µg 1 nmol

10,000 100 pmol or 6 × 1013 molecules 10 µg

50,000 20 pmol or 1.2 × 1013 molecules 50 µg

100,000 10 pmol or 6 × 1012 molecules 100 µg

150,000 6.7 pmol or 4 × 1012 molecules 150 µg

concentration of protein absorbance of protein Protein A280 units for 1 mg/ml

IgG 1.35

IgM 1.2

IgA 1.3

protein A 0.17

avidin 1.5

streptavidin 3.4

bovine serum albumin 0.7

Calculationsmolarity of a solution =

mass of solute (g)

[molecular mass of solute (g/mol)] × (L of solution)

molecular mass of a ds DNA fragment = (number of bp) × (660 Da/bp)

moles of ends of a ds DNA fragment =

2 × (g of DNA)/[molecular mass of DNA (Da)]

or = 2 × (g of DNA)/[(number of bp) × (660 Da/bp)]

moles of ends generated by a restriction endonuclease

circular DNA: 2 × (moles of DNA) × (number of sites)

linear DNA: 2 × (moles of DNA) × (number of sites)

+ 2 (original ends)

Units of radioactivity 1 µCi = 2.2 × 106 disintegrations per minute = 3.7 × 104 becquerels

1 becquerel = 1 disintegration per second

Half-life of common radioisotopesRadioisotope Half-life

Carbon-14 (14C) 5,730 years

Iodine-125 (125I) 60 days

Phosphorus-32 (32P) 14.3 days

Sulfur-35 (35S) 87.4 days

Tritium (3H) 12.4 years

Metric prefixesM = mega = 106 n = nano = 10–9

k = kilo = 103 p = pico = 10–12

m = milli = 10–3 f = femto = 10–15

µ = micro = 10–6 a = atto = 10–18

Abbreviationsds . . . . . . double-stranded (as in ds DNA)

ss . . . . . . . single-stranded (as in ss DNA)

bp . . . . . . base pair

kb . . . . . . kilobase: 1,000 bases or base pairs, as appropriate

Da . . . . . . dalton, the unit of molecular mass

mol . . . . . mole

M . . . . . . . molarity, moles of solute per liter of solution




Genetic Code and Amino Acid Abbreviations

U

C

A

G

U

UUU PhenylalanineUUC (Phe, F)

UUA LeucineUUG (Leu, L)

CUUCUC LeucineCUA (Leu, L)CUG

AUU IsoleucineAUC (Ile, I)AUA

AUG Methionine (Met, M)

GUUGUC ValineGUA (Val, V)GUG

}}

}

A

UAU TyrosineUAC (Tyr, Y)

UAA STOPUAG

CAU HistidineCAC (His, H)

CAA GlutamineCAG (Gln, Q)

AAU AsparagineAAC (Asn, N)

AAA LysineAAG (Lys, K)

GAU Aspartic acidGAC (Asp, D)

GAA Glutamic acidGAG (Glu, E)

C

UCUUCC SerineUCA (Ser, S)UCG

CCUCCC ProlineCCA (Pro, P)CCG

ACUACC ThreonineACA (Thr, T)ACG

GCUGCC AlanineGCA (Ala, A)GCG

G

UGU CysteineUGC (Cys, C)

UGA STOP

UGG Tryptophan (Trp, W)

CGUCGC ArginineCGA (Arg, R)CGG

AGU SerineAGC (Ser, S)

AGA ArginineAGG (Arg, R)

GGUGGC GlycineGGA (Gly, G)GGG

} }}

} } }

} }}

}}}}

}}

}}

}

}Firs

t pos

ition

Second position


234


Notes:


Appendix IITerms and Conditions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 236

Trademark Information . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 238

Appendix II Trademark Information

236


Terms and ConditionsThe following is an abbreviated description of our General Terms and Conditions. The General Terms and Conditions are included with or on Invitrogen invoices or pack slips and control the sale of products by Invitrogen. In the event of any inconsistency between this catalogue and the General Terms and Conditions, then the General Terms and Conditions shall control. Any customer who does not have a copy of our General Terms and Conditions may obtain a copy by contacting Customer Service. Our General Terms and Conditions are also available on our website at www.invitrogen.com. In addition, customers outside the U.S. should contact their local distributor for a copy of the General Terms and Conditions applicable for their local jurisdiction.

Quick DeliveryIf you require special delivery, please request this when placing your order. We will make every attempt to get your order to you when you need it. As appropriate, we will arrange and bill for this service accordingly.

Changes or CancellationsIf your catalogue order has not been shipped, you may initiate a request over the phone to change the order or cancel it. You’ll need to confirm your request in writing, as well, and may do so by using the appropriate toll-free ordering fax lines.

ShippingUnless we agree otherwise, freight terms are FOB Life Technologiestm

shipping point with freight charges added to your invoice as a separate item. We do not require written confirmation of orders, except for standing, blanket, and change orders. For these exceptions, please clearly mark “Confirming —Do Not Duplicate” on your written confirmation. We take great care to choose the most appropriate shipping methods for each catalogue order based on feedback from our customers. We ensure the accuracy of each order, and we schedule each shipment to arrive at your destination during normal business hours. We reserve the right to make delivery in installments, all such installments to be separately invoiced and paid for when due per invoice. If your complete order does not arrive promptly and in optimal condition, please notify a Customer Service Representative.

Special Shipping Conditions or CarriersWe use air freight and surface carriers to ensure prompt and cost-effective delivery of your order. If you require other arrangements, just tell us when you place your order. We ship at room temperature or on dry ice or wet ice, as detailed in the product descriptions in this catalogue.

Damaged or Incomplete ShipmentIf your shipment is visibly damaged, accept the shipment and retain all shipping documents, cartons, and packing materials for inspection. Note on the waybill any visible damage at the time of receipt and report the damage to a Customer Service Representative as soon as possible. You should also call if you find concealed damage or the shipment is incomplete.

ReturnsIf a Life Technologiestm catalogue product does not conform to its description in this catalogue or specifications in the product insert sheet, let us know. We will credit your account or exchange the product at our option. To return any catalogue product, you must first obtain a return goods authorization (RGA) number. Please contact your Customer Service Representative for instructions. A restocking fee of twenty-five percent (25%) of the amount invoiced will be charged on the return of any product for any reason other than under our warranty described in this catalogue.

Our WarrantyLife Technologiestm sells products in good faith, believing that the products we deliver to you, the original purchaser of our products, will match the description you read in our catalogue and the specifications in the product insert sheet. Below is an abbreviated description of our warranty. The complete warranty statement is located on the reverse of Life Technologiestm invoices or packing lists. In summary, if the product is defective or nonconforming, we will either replace the product or refund the purchase price of the product, provided that: (i) if the problem results from incomplete delivery, you advise us within

three (3) days of receipt;(ii) you did not cause the problem by altering or misusing the product,

or the like;(iii) you are the original purchaser of the product; and you advised

Customer Service by calling the appropriate telephone number within the specified time limits, and complied with the instructions received from the Customer Service Representative.

OUR WARRANTY IS LIMITED, AND UNDER NO CIRCUMSTANCES WILL WE BE LIABLE FOR ANY INDIRECT, SPECIAL, INCIDENTAL, CONSEQUENTIAL, OR EXEMPLARY DAMAGES IN EXCESS OF THE PURCHASE PRICE PAID. All terms and conditions, including price, are subject to change without notice, unless otherwise specified in our Quotation.

Acceptance of ProductsUnless we receive written notification from you within three (3) days after receipt, your acceptance of the products shipped by us constitutes your assent to the general terms and conditions under which we offer these materials for sale.

Appendix IIgibco® cell culture catalogue



Patent Warranty and DisclaimerWe warrant to you, our customers, that the manufacture and sale of the products manufactured by or for us without reliance upon instructions, specifications, or other directions provided by you to our knowledge will not infringe the claims of any patent, trademark or copyright of any third party. We do not warrant that the manufacture and sale by us of products manufactured in reliance upon instructions, specifications, or other directions provided by you or your use or resale of the products will not infringe the claims of any patent, trademark, or copyright of any third party.

Authorized Use of ProductsExcept as otherwise agreed in writing by an authorized representative of Invitrogen, the purchase of products only conveys to you the non-transferable right to use the products purchased in compliance with the applicable intended use statement, limited use statement, or limited use label license, if any, in our catalogues or on the label or other documentation accompanying the products. Unless otherwise expressly indicated in our catalogues or on the label or other documentation accompanying the products, the products are intended for research use only and are not to be used for any other purposes including, but not limited to, unauthorized commercial purposes, in vitro diagnostic purposes, ex vivo or in vivo therapeutic purposes, investigational use, in foods, drugs or cosmetics of any kind, or for consumption by or use in connection with or administration or application to humans or animals. You acknowledge that the products have not been tested by or for us for safety or efficacy, unless expressly stated in our catalogues or on the label or other documentation accompanying the products. Without limiting the foregoing restrictions, you warrant to us that should you use or sell the products for any use other than research, you shall conduct all necessary tests, comply with all applicable regulatory requirements, issue all appropriate warnings and information to subsequent purchasers and/or users and be responsible for obtaining any required Intellectual Property rights. Please contact the Company’s Licensing Development department through any of the contacts listed on the back of this catalogue to obtain information regarding use of products or their components for purposes other than as authorized above.

Please Read the LabelPlease note the expiration dates, if any, and follow the proper usage and storage instructions provided on the label or product profile for each product. We cannot be liable for a product after its expiration date or for one that has been misused or has become unusable due to improper storage, handling, or label alterations. In certain circumstances, the label, product profile, or Material Safety Data Sheet (MSDS) contain important information, instructions, or warnings that were not included in this catalogue. Therefore, it is important to review these items carefully.

Note HazardsPlease read the label for information about product hazards. All Life Technologiestm products should be handled only by qualified persons trained in laboratory safety procedures. Some products may present flammable, corrosive, acute and chronic, toxic, reactive, or other hazards. Product-specific hazard warnings are included in this catalog, in product literature, on product labels, and in MSDSs where applicable. All possible hazards of many products may not be known at this time: the absence of a warning must not be interpreted as an indication that the product is safe. Please report any information of new effects (not already described in literature, on labels, or on MSDSs) to Invitrogen Technical Support. Always use good laboratory procedures when handling any products.

Product Labeling and UsageThe products listed in the catalogue are for research use only, unless otherwise stated. Products labeled “For in vitro diagnostic use” are medical devices and as such are required to comply with the Food and Drug Administration Good Manufacturing Practices known as The Quality System Regulation (Code of Federal Regulations Title 21, Part 820). These products may also be used in laboratory research applications. These products are not qualified nor intended for any animal or human therapeutic uses. Inquiries into the use of Life Technologiestm products are welcome and should be directed to Life Technologiestm Technical Support. Requests will be considered on a case-by-case basis as appropriate under applicable government regulations.

By placing an order, you agree to the following:The purchaser is responsible for compliance with Food and Drug Administration regulations, all state laws, and any other federal or local laws or regulations governing the use of these materials. Invitrogen recommends that the buyer or other user adhere to NIH guidelines published in the Federal Register, Volume 41, No. 131, July 7, 1976, and any amendments thereto. Invitrogen disclaims any and all responsibility for any injury or damage which may occur from the use of these materials, except as provided in the express warranty set forth above.

Appendix II Trademark Information

238


Trademark InformationLife Technologiestm has achieved recognition of its trademarks worldwide. Please respect our trademarks; we will vigorously protect their proper usage. All names containing the designation ® are registered with the U.S. Patent and Trademark Office. Such trademarks may or may not be registered in other countries.

Trademarks of Life Technologiestm and its affiliated companies

293fectin™ ■

AIM V® ■

AlbuMAX® ■

AlgiMatrix™ ■

AmnioMAX™ ■

Advanced Granulation Technology™ (AGT™) ■

Bac-N-Blue™ ■

Bac-to-Bac® ■

BaculoDirect™ ■

BLOCK-iT™ ■

Cellfectin® ■

EpiLife® ■

Express Five® ■

E-Z Hold™ ■

EZPassage™ ■

FoamAway™ ■

FreeStyle™ ■

Gateway® ■

Geltrex™ ■

Geneticin® ■

gibco ® ■

GlutaMAX™ ■

PB-MAX™ ■

PD-Direct™ ■

pcDNA™ ■

PLUS™ ■

pOptiVEC™ ■

Recovery™ ■

Revolution™ ■

rProtease™ ■

SimCell™ ■

Sf-900™ ■

Stak Pack™ ■

StemPro ■ ®SynerGy™ ■

Synth-a-Freeze® ■

TA Cloning® ■

Tech-Line™ ■

Tech OnLine™ ■

TOPO® ■

TrypLE™ ■

ViraPower™ ■

Voyager™ ■

High Five™ ■

iMATCH™ ■

InsectSelect™ ■

Invitrogen™ ■

KaryoMAX® ■

KnocKout ■ ™Lipofectamine™ ■

MarrowMAX™ ■

MediaExpress ■ SM

MesenPRO™ ■

MesenPRO RS™ ■

Neurobasal™ ■

Oligofectamine™ ■

One Shot® ■

One Shot™ ■

OptiCHO™ ■

OptiMab® ■

Opti-MEM® ■

OptiPRO™ ■

pAd/BLOCK-iT™ ■

pAd/PL-DEST™ ■

Other companies’ trademarks

C-flex® (Consolidated Polymer Technologies) ■

Colcemid® (Ciba Ltd.) ■

Cellbag® (Wave Biotech) ■

Corning™ (Corning Inc.) ■

Fungizone® (E.R. Squibb & Sons, L.L.C.) ■

Matrigel™ (Becton Dickinson) ■

PER.C6® (Crucell Holland B.V.) ■

Pluronic® (BASF Corporation) ■

Solvay™ (Solvay SA) ■

Tween® (ICI Americas, Inc.) ■

ViCell® Cell Counter (Beckman Coulter) ■

Zeocin™ (CAYLA) ■

Lab Armor™ ■


Index

Alphabetical Index . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 240

Catalogue Number Index . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 246

Alphabetical Index

Page Page Page

240


250X Cholesterol Lipid Concentrate . . . . . .106, 163, 166

293 Expression Products . . . . . . . . . . . . . . . .93

293 Cells . . . . . . . . . . . . . . . . . . . . . 177

293 SFM II . . . . . . . . . . . . . . . . . . . . . .93

CD 293 AGT™ . . . . . . . . . . . . . . . . . . . .93

FreeStyle™ 293 Expression Medium . . . . . . . . . .93

FreeStyle™ 293 Expression System . . . . . . . . . .94

293fectin™ Transfection Reagent . . . . . . . . . . . 133

2-Mercaptoethanol (1,000X), liquid . . . . . . . . . . 168

3D Matrices and Scaffolds . . . . . . . . . . . . . . 88

AlgiMatrix® 3D Culture System . . . . . . . . . . . . 6

4% Agarose Gel . . . . . . . . . . . . . . . . . . . 103

AACK Lysing Buffer . . . . . . . . . . . . . . . . . . 149

Activin A . . . . . . . . . . . . . . . . . . . . . . .14

Actinomycin D . . . . . . . . . . . . . . . . . . . 150

Adaptation of Cell Cultures to a Serum-free Medium . . 224

Adaptation of Cell Cultures to an Alternative Serum . . 225

Adaptation of Cells from Monolayer to Suspension Culture . . . . . . . . . . . . . . . . 225

Advanced DMEM . . . . . . . . . . . . . . . . . . .30

Advanced DMEM/F-12 . . . . . . . . . . . . . . . . .33

Advanced MEM (Minimum Essential Medium), with L-gluatmine . . . . . . . . . . . . . . . . . .40

Advanced RPMI Medium 1640 (1X), liquid . . . . . . . .45

Adenovirus Expression Medium (AEM). . . . . . . . . .96

AIM V® Medium CTS™ . . . . . . . . . . . . . . . . . 3

AIM V® Medium (research grade) . . . . . . . 112, 117

AIM V® Medium (therapeutic grade) . . . . . . 112, 118

AlbuMAX® . . . . . . . . . . . . . . . . . . . . . . 163

AlgiMatrix® 3D Culture System . . . . . . . . . . . . . 6

Amino Acids . . . . . . . . . . . . . . . . . . . . 149

GlutaMAX™-I Supplement . . . . . . . . . . . . . 149

L-Glutamine . . . . . . . . . . . . . . . . . . . . 149

L-Glutamine-200 mM . . . . . . . . . . . . . . . 149

MEM Amino Acids Solution . . . . . . . . . . . . . 149

AmnioMAX™ . . . . . . . . . . . . . . . . . . . . 127

Antibiotic-Antimycotic (100X), liquid . . . . . . . . . 129

Antibiotics and Antimycotics . . . . . . . . . . . . . 150

Actinomycin D . . . . . . . . . . . . . . . . . . 150

Ampicillin . . . . . . . . . . . . . . . . . . . . . 150

Antibiotic-Antimycotic . . . . . . . . . . . . . 129, 150

Blasticidin S HCl . . . . . . . . . . . . . . . . . . 151

Puromycin . . . . . . . . . . . . . . . . . . . . 151

Carbenicillin . . . . . . . . . . . . . . . . . . . . 151

Geneticin® Selective Antibiotic . . . . . . . . . . . 152

Gentamicin Reagent . . . . . . . . . . . . . . . . 153

Hygromycin B . . . . . . . . . . . . . . . . . . . 153

Kanamycin Sulfate . . . . . . . . . . . . . . . . . 153

Mycophenolic Acid . . . . . . . . . . . . . . . . 154

Neomycin Sulfate . . . . . . . . . . . . . . . . . 154

Penicillin-Streptomycin . . . . . . . . . . . . . . 154

Penicillin-Streptomycin-Glutamine . . . . . . . . . 154

Penicillin-Streptomycin-Neomycin (PSN) . . . . . . 155

Polymyxin B Sulfate . . . . . . . . . . . . . . . . 155

Streptomycin Sulfate . . . . . . . . . . . . . . . . 155

Zeocin™ . . . . . . . . . . . . . . . . . . . . . 155

Antifoaming and Defoaming Reagents . . . . . . . . 156

FoamAway™ Irradiated . . . . . . . . . . . . . . . 156

FoamAway™ Irradiated AOF . . . . . . . . . . . . 156

Attachment Factors . . . . . . . . . . . . . . . . . 162

Fibronectin, Human Plasma . . . . . . . . . . . . 162

Natural Mouse Laminin . . . . . . . . . . . . . . 162

BBags . . . . . . . . . . . . . . . . . . . . . . . . 206

Gibco® Universal Bags . . . . . . . . . . . . . . . 206

Prefilled Wave bag . . . . . . . . . . . . . . . . . 207

Balanced Salt Solutions . . . . . . . . . . . . . . . 138

Balanced Salt Solutions Preparation . . . . . . . . . . 142

Balanced Salt Solutions Formulations . . . . . . . . . 143

Basal Medium Eagle (BME) . . . . . . . . . . . . . . .28

BGJb Medium (Fitton-Jackson Modification) . . . . . . .28

Biological Buffers . . . . . . . . . . . . . . . . . . 157

HEPES Buffer Solution (1 M) . . . . . . . . . . . . 157

HEPES Ultrapure . . . . . . . . . . . . . . . . . . 157

Sodium Bicarbonate Solution, 7.5% . . . . . . . . . 157

Blasticidin S HCI . . . . . . . . . . . . . . . . . 123, 151

BMP-4 . . . . . . . . . . . . . . . . . . . . . . . .14

BMP-7 . . . . . . . . . . . . . . . . . . . . . . . .14

Bovine and Rat Tail Collagen . . . . . . . . . . . . . . 5

Bovine Sera . . . . . . . . . . . . . . . . . . . . . 191

Newborn Calf Sera . . . . . . . . . . . . . . . . . 191

Bovine Serum, heat-inactivated . . . . . . . . . . . 191

Donor Bovine Serum . . . . . . . . . . . . . . . . 191

Donor Bovine Serum, with iron . . . . . . . . . . . 191

Brinster’s BMOC-3 Medium . . . . . . . . . . . . . . .29

Bronchial Epithelial Products . . . . . . . . . . . . . 109

Buffer Systems for Cell Culture . . . . . . . . . . . . 219

CCarbenicillin, Disodium Salt . . . . . . . . . . . . . . 151

CD DG44 Medium . . . . . . . . . . . . . . . . . . 180

CD Hybridoma Medium (1X), liquid . . . . . . . . . . 106

CD Hybridoma AGT™ . . . . . . . . . . . . . . . . . 106

CD OptiCHO™ Medium . . . . . . . . . . . . . . . 180

Cell Culture basics . . . . . . . . . . . . . . . . . . 210

Cell Culture Products Overview . . . . . . . . . . . . . ii

Cell Culture Reagents . . . . . . . . . . . . . . . . 148

Other Cell Culture Reagents . . . . . . . . . . . . 168

Cell Culture Reagent Formulations . . . . . . . . . 169

Cell Dissociation Products . . . . . . . . . . . . . . 158

Cell Dissociation Buffers . . . . . . . . . . . . . . 158

Collagenase, lyophilized . . . . . . . . . . . . . . 158

Collagenase Type I . . . . . . . . . . . . . . . . . 158

Collagenase Type II . . . . . . . . . . . . . . . . 158

Collagenase Type IV . . . . . . . . . . . . . . . . 158

Dispase . . . . . . . . . . . . . . . . . . . . . . 158

TrypLE™ Select Stable Trypsin Replacement Enzyme (1X), liquid . . . . . . . . . 159

TrypLE™ Express, Stable Trypsin Replacement Enzyme (1X), liquid . . . . . . . . . 160

Trypsin, 0.25% (1X), liquid . . . . . . . . . . . . . 160

Trypsin, 2.5% (10X), liquid . . . . . . . . . . . . . 160

Trypsin-EDTA (0.05% Trypsin, EDTA•4Na) (1X), liquid . 160

Trypsin-EDTA (0.5% Trypsin, EDTA•4Na) (10 X), liquid . 160

Trypsin-EDTA (0.25% Trypsin, EDTA•4Na) (1X), liquid . 160

Trypsin (1:250), powder . . . . . . . . . . . . . . 161

Trypsin Inhibitor, soybean . . . . . . . . . . . . . 161

Versene 1:5,000 (1X), liquid

Cellfectin® Reagent . . . . . . . . . . . . . . . . . 103

Cellfectin® II Transfection Reagent . . . . . . . . . . . 133

Cell Line Development Kits . . . . . . . . . . . . . . 180

OptiCHO™ Express Kit . . . . . . . . . . . . . . . 180

OptiCHO™ Antibody Express Kit . . . . . . . . . . 181

Cell Therapy Systems (CTS™) . . . . . . . . . . . . . . 2

CELLstart™ Cell Attachment/Expansion Substrate . . . . 4

Cellular Studies . . . . . . . . . . . . . . . . . . . 109

Bronchial Epithelial Products . . . . . . . . . . . . 109

Dermatology Products . . . . . . . . . . . . . . . 110

Hematology-Oncology Products . . . . . . . . . . 110

Hematopoiesis Products . . . . . . . . . . . . . . 113

Hepatocyte Products . . . . . . . . . . . . . . . 113

Human Mammary Epithelial Products . . . . . . . . 114

Neurobiology Products . . . . . . . . . . . . . . 114

Chicken Serum . . . . . . . . . . . . . . . . . . . 192

CHO-S Cells . . . . . . . . . . . . . . . . . . . . . 178

CMRL Medium . . . . . . . . . . . . . . . . . . . .29

CMRL Medium, 1066 (1X) + (10), liquid . . . . . . . .29

CO2-Independent Medium . . . . . . . . . . . . . . .29

CO2-Independent Medium (1X), liquid . . . . . . . .29

Collagenase, lyophilized (Clostridiopeptidase A) . . . . 130

Collagen 1 . . . . . . . . . . . . . . . . . . . . . . 5

Conversion Factors, Units, and Abbreviations . . . . . . 232

Cryopreservation of Mammalian Cells . . . . . . . . . 227

Thawing of Cryopreserved Cells . . . . . . . . . . 227

Cryopreservation Medium . . . . . . . . . . . . . . .29

CTS™ cytokines and growth factors . . . . . . . . . . . 3

Cytogenetics Products . . . . . . . . . . . . . . . . 126

MarrowMAX™ . . . . . . . . . . . . . . . . . . . 126

MarrowMAX™ Bone Marrow Medium . . . . . . . . 126

AmnioMAX™ . . . . . . . . . . . . . . . . . . . 127

AmnioMAX™-II Complete Medium . . . . . . . . . 127

AmnioMAX™-C100 Products . . . . . . . . . . . . 127

KaryoMAX® . . . . . . . . . . . . . . . . . . . . 127


Page Page Page

Alphabetical Index



PB-MAX™ Karyotyping Medium . . . . . . . . . . . 127

KaryoMAX® Colcemid™ Solution, liquid (10 µg/ml), in HBSS . . . . . . . . . . . . 127

Cytogenetics: Related Products . . . . . . . . . . . . 128

Gurr Buffer Tablets (pH 6.8) . . . . . . . . . . . . . 128

Phytohemagglutinin (M Form) (PHA), liquid . . . . . 128

Potassium Chloride Solution, 0.075 M . . . . . . . . 128

DDecontaminating Cultures with Antibiotics

and Antimycotics . . . . . . . . . . . . . . . . . 231

Use of Antibiotics and Antimycotics . . . . . . . . . 231

Defined Keratinocyte-SFM System . . . . . . . . . . 110

Dissociation of Cells from Primary Tissue . . . . . . . . 228

Dissociation of Cells from Culture Vessels . . . . . . . 229

Distilled Water . . . . . . . . . . . . . . . . . . . 161

Dermatology Products . . . . . . . . . . . . . . . . 110

Keratinocyte Starter Kit . . . . . . . . . . . . . . . 110

Keratinocyte-SFM (1X), liquid . . . . . . . . . . . . 110

Keratinocyte-SFM (1X), liquid (without CaCl2) . . . . 110

DG44 Cells . . . . . . . . . . . . . . . . . . . . . 178

D-Glucose (dextrose) . . . . . . . . . . . . . . . . . 168

D.Mel-2 Cells . . . . . . . . . . . . . . . . . . . . 174

DPBS CTS™ . . . . . . . . . . . . . . . . . . . . . . 3

Dulbecco’s Modified Eagle Media (DMEM) . . . . . . . .30

Advanced DMEM (1X), liquid . . . . . . . . . . . . .30

DMEM (1X), liquid (high glucose) . . . . . . . . . . .30

DMEM (1X), liquid (low glucose) . . . . . . . . . . .32

DMEM (1X), liquid (no glucose) . . . . . . . . . . . .32

DMEM, powder (low glucose) . . . . . . . . . . . .32

Advanced DMEM/F-12 (1X), liquid, 1:1 . . . . . . . . .33

DMEM/F-12 (1X), liquid, 1:1 . . . . . . . . . . . . . .33

Dulbecco’s Phosphate-Buffered Salines . . . . . . . . 138

Dulbecco’s Phosphate-Buffered Saline (DPBS)

(1X), liquid . . . . . . . . . . . . . . . . . . . . 138

Dulbecco’s Phosphate-Buffered Saline (DPBS)

(10X), liquid . . . . . . . . . . . . . . . . . . . . 139

Dulbecco’s Phosphate-Buffered Saline (DPBS),

powder . . . . . . . . . . . . . . . . . . . . . . 139

Dynabeads® CD3/CD28 CTS™ . . . . . . . . . . . . . 3

DynaMag™ CTS™ . . . . . . . . . . . . . . . . . . . 3

EEarle’s Balanced Salt Solution . . . . . . . . . . . . . 139

Earle’s Balanced Salt Solution (EBSS) (1X), liquid . . . 139

Expression Products . . . . . . . . . . . . . . . . . 89

CHO Expression Products . . . . . . . . . . . . . .89

Customized CHO Medium Products . . . . . . . . . .90

DG44 Expression Products . . . . . . . . . . . . . .91

293 Expression Products . . . . . . . . . . . . . . .93

Virus Production Medium Products . . . . . . . . . .94

Viral Expression Products . . . . . . . . . . . . . . .97

Insect Cell Expression Products . . . . . . . . . . . .98

Customized Insect Medium Products . . . . . . . . 101

Insect Medium Supplements . . . . . . . . . . . . 103

Hybridoma and Monoclonal Antibody Products . . . 106

Customized Hybridoma Medium Products . . . . . 107

E-Z Hold™ Plastic Bottles . . . . . . . . . . . . . . . .49

FF-10 Nutrient Mixtures . . . . . . . . . . . . . . . . .34

F-10 Nutrient Mixture (Ham) (1X), liquid . . . . . . . .34

F-12 Nutrient Mixtures . . . . . . . . . . . . . . . . .34

F-12 Nutrient Mixture (Ham) (1X), liquid . . . . . . . .34

F-12 Nutrient Mixture (Ham), powder . . . . . . . . .34

F-12K Nutrient Mixture,

Kaighn’s Modification (1X), liquid . . . . . . . . . . .34

FBS Selection Guide . . . . . . . . . . . . . . . . . 187

FBS Quality Control Test Comparison . . . . . . . . . 193

Other Sera Quality Control Test Comparison . . . . . 194

Fetal Bovine Sera (FBS) and other Sera for Cell Culture . 186

General Animal Sera Information . . . . . . . . . . 186

The iMATCH™ MPA Sera Lot Matching Tool . . . . . . 186

Fetal Bovine Sera (FBS) . . . . . . . . . . . . . . . . 189

qualified . . . . . . . . . . . . . . . . . . . . . 188

qualified, heat-inactivated . . . . . . . . . . . . . 188

qualified, gamma irradiated . . . . . . . . . . . . 189

dialyzed . . . . . . . . . . . . . . . . . . . . . 189

charcoal stripped . . . . . . . . . . . . . . . . . 189

ES cell qualified . . . . . . . . . . . . . . . . . . 189

MSC qualified . . . . . . . . . . . . . . . . . . . 190

ultra-low IgG FBS . . . . . . . . . . . . . . . . . 190

FGF-basic . . . . . . . . . . . . . . . . . . . . . . .12

Fibronectin, Human Plasma . . . . . . . . . . . . . . 162

Final Product Quality Control . . . . . . . . . . . . 196

FoamAway™ Irradiated . . . . . . . . . . . . . . . . 156

FoamAway™ Irradiated AOF . . . . . . . . . . . . . 156

Formulas and Formula Weights for Commonly Used Medium Components . . . . . . . . . . . . . . . 216

FreeStyle™ MAX Reagent . . . . . . . . . . . . . . . 132

FreeStyle™ 293 Expression Medium . . . . . . . . . . 177

Fungizone™ Antimycotic, liquid . . . . . . . . . . . . 129

GGeltrex™ Reduced Growth Factor Basement Membrane

Matrix . . . . . . . . . . . . . . . . . . . . . . . 5

Genetic Code and Amino Acid Abbreviations . . . . . 233

Geneticin® Selective Antibiotic . . . . . . . . . . . . 122

Geneticin® Selective Antibiotic, liquid . . . . . . . . . 152

Gentamicin Reagent Solution (10 mg/ml), liquid . . . . 129

Gentamicin Reagent Solution, liquid . . . . . . . . . 153

Geneticin® Selective Antibiotic, powder . . . . . . . . 153

Gibco® Cell Therapy Systems (CTS™) . . . . . . . . . . 2

CELLstart™ Cell Attachment/Expansion Substrate . . . 4

Geltrex™ Reduced Growth Factor Basement Membrane Matrix . . . . . . . . . . . . . . . . . . . . . . 5

Bovine and Rat Tail Collagen I . . . . . . . . . . . . . 5

Gibco® CTS™ Immunotherapy products . . . . . . . . . 3

AIM V® Medium CTS™—Therapeutic-grade, serum-free cell expansion medium . . . . . . . . . 3

OpTmizer™ CTS™ T-Cell Expansion SFM . . . . . . . . 3

CTS™ cytokines and growth factors . . . . . . . . . . 3

DPBS CTS™ . . . . . . . . . . . . . . . . . . . . . 3

Dynabeads® CD3/CD28 CTS™ . . . . . . . . . . . . 3

DynaMag™ CTS™ . . . . . . . . . . . . . . . . . . 3

Interleukin 4 CTS™ . . . . . . . . . . . . . . . . . . 3

Gibco® CTS™ products for stem cells . . . . . . . . . . 4

Gibco® LIF Recombinant . . . . . . . . . . . . . . . .13

Gibco® Recombinant Growth Factors for use in stem cell applications . . . . . . . . . . . . . . . . . . . . .14

Gibco® Recombinant human FGF-basic . . . . . . . . .12

Gibco® Universal Bags . . . . . . . . . . . . . . . . 204

Prefilled Wave Bag . . . . . . . . . . . . . . . . . 207

Glasgow Minimum Essential Medium . . . . . . . . . .35

Glasgow Minimum Essential Medium . . . . . . . . .35

(G-MEM, BHK-21) (1X), liquid

GlutaMAX™ Media at a glance . . . . . . . . . . . . .26

GlutaMAX™-I Supplement . . . . . . . . . .123, 130, 149

Goat Serum . . . . . . . . . . . . . . . . . . . . . 192

Grace’s Insect Cell Culture Media . . . . . . . . . . . .35

Grace’s Insect Cell Culture Medium,

supplemented (1X), liquid . . . . . . . . . . . . . .35


supplemented (2X), liquid . . . . . . . . . . . . . .35


unsupplemented (1X), liquid . . . . . . . . . . . . .35

Grace’s Insect Cell Culture Medium, powder . . . . . .35

Growth factors, Cytokines and Chemokines with high bioactivity . . . . . . . . . . . . . . . . . . . . .10

Growth Supplements . . . . . . . . . . . . . . . . 163

250X Cholesterol Lipid Concentrate . . . . . . . . . 163

Chemically Defined Lipid Concentrate . . . . . . . 163

AlbuMAX® I . . . . . . . . . . . . . . . . . . . . 163

AlbuMAX® II . . . . . . . . . . . . . . . . . . . . 163

Bovine Albumin Fraction V Solution (7.5%) . . . . . . 163

B-27 Serum-free Supplements . . . . . . . . . . . 164

G-5 Supplement (100X), liquid . . . . . . . . . . . 164

Insulin, Human Recombinant, Zinc . . . . . . . . . 164

Insulin-Transferrin-Selenium Supplements (ITS) . . . 164

Insulin-Transferrin-Selenium-A Supplement (100X) . . 164

Insulin-Transferrin-Selenium-X Supplement (100X) . . 165

Lactalbumin Hydrolysate, powder (extra soluble). . . 165

N-2 Supplement (100X), liquid . . . . . . . . . . . 165

Transferrin, Bovine (APO form), lyophilized . . . . . . 165

Transferrin, Bovine (holo form), lyophilized . . . . . . 165

Transferrin, Bovine (partially iron loaded), lyophilized . 165

Alphabetical Index

Page Page Page

242


HHanks’ Balanced Salt Solutions . . . . . . . . . . . . 140

Hanks’ Balanced Salt Solution (HBSS) (1X), liquid . . . 140

Hanks’ Balanced Salt Solution (HBSS) (10X), liquid . . 140

HAT Supplement (50X), liquid . . . . . . . . . . . . . 107

Gibco® Hepatocytes . . . . . . . . . . . . . . . . . 182

Hematopoiesis Products . . . . . . . . . . . . . . . 113

StemPro®-34 SFM (1X), liquid . . . . . . . . . . . . 113

Macrophage-SFM (1X), liquid . . . . . . . . . . . . 113

Hepatocyte Products . . . . . . . . . . . . . . . . . 113

Hepatozyme-SFM (1X), liquid . . . . . . . . . . . . 113

Liver Digest Medium (1X), liquid . . . . . . . . . . 113

Liver Perfusion Medium (1X), liquid . . . . . . . . . 113

Hepatocyte Wash Medium . . . . . . . . . . . . . 113

HEPES Buffer Solution (1 M) . . . . . . . . . . . . . . 157

HEPES Media at a glance . . . . . . . . . . . . . . . .27

HEPES Ultrapure . . . . . . . . . . . . . . . . . . . 157

High Five™ Cells . . . . . . . . . . . . . . . . . . . 174

Horse Sera . . . . . . . . . . . . . . . . . . . . . 192

Horse Serum . . . . . . . . . . . . . . . . . . . 192

Horse Serum, heat-inactivated . . . . . . . . . . . 192

Human Mammary Epithelial Products . . . . . . . . . 114

HuMEC Medium . . . . . . . . . . . . . . . . . . 114

Human proteins . . . . . . . . . . . . . . . . . . . .15

HT Supplement (100X), liquid . . . . . . . . . . . . . 107

Hybridoma and Monoclonal Antibody Products . . . . 106

Hybridoma Reagents . . . . . . . . . . . . . . . . 166

250X Cholesterol Lipid Concentrate . . . . . . . . . 166


HAT Supplement (50X), liquid. . . . . . . . . . . . 166

HT Supplement (100X), liquid . . . . . . . . . . . . 166

Hybridoma-SFM (1X), liquid . . . . . . . . . . . . . . 106

Hygromycin B . . . . . . . . . . . . . . . . . . 123, 153

IIL-17F . . . . . . . . . . . . . . . . . . . . . . . .15

iMATCH™ MPA Sera Lot Matching Tool . . . . . . . . 186

Improved MEM Zn++ Option (Richter’s Modification) . . .36

Insect Cells . . . . . . . . . . . . . . . . . . . . . 174

D.Mel-2 Cells . . . . . . . . . . . . . . . . . . . 174

High Five™ Cells . . . . . . . . . . . . . . . . . . 174

Sf9 Cells . . . . . . . . . . . . . . . . . . . . . 175

Sf21 Cells . . . . . . . . . . . . . . . . . . . . . 175

Insect Cell Expression Products . . . . . . . . . . . . .98

Sf-900™ III SFM . . . . . . . . . . . . . . . . . . .99

Sf-900™ II SFM . . . . . . . . . . . . . . . . . . . .99

Insect Medium Supplements . . . . . . . . . . . 103, 167

4% Agarose Gel . . . . . . . . . . . . . . . . . . 167


Lactalbumin Hydrolysate, powder (extra soluble). . . 167

Pluronic® F-68, 10% (100X) . . . . . . . . . . . . . 167

Yeastolate Ultrafiltrate (50X), liquid . . . . . . . . . 167

Interleukin 4 CTS™ . . . . . . . . . . . . . . . . . . . 3

IPL-41 Insect Medium . . . . . . . . . . . . . . . . .36

Iscove’s Modified Dulbecco’s Media . . . . . . . . . . .36

Iscove’s Modified Dulbecco’s Medium

(IMDM) (1X), liquid . . . . . . . . . . . . . . . . . .36

Iscove’s Modified Dulbecco’s Medium (IMDM),

powder . . . . . . . . . . . . . . . . . . . . . . .36

KKanamycin Sulfate . . . . . . . . . . . . . . . . . . 153

Kanamycin Sulfate (100X), liquid . . . . . . . . . . . 153

KaryoMAX® . . . . . . . . . . . . . . . . . . . . . 127

KnockOut™ Serum Replacement . . . . . . . . . . . 120

KnockOut™ DMEM . . . . . . . . . . . . . . . . . . 121

LLab Armor™ Beads . . . . . . . . . . . . . . . . . . 208

Lamb Serum . . . . . . . . . . . . . . . . . . . . 192

Leibovitz’s L-15 Media . . . . . . . . . . . . . . . . .37

L-Glutamine . . . . . . . . . . . . . . . . . . . . . 149

L-Glutamine-200 mM, liquid . . . . . . . . . . . . . 149

LHC Medium (1X), liquid . . . . . . . . . . . . . . . 109

LIF Recombinant . . . . . . . . . . . . . . . . . . .13

Lipofectamine™ Transfection Reagent . . . . . . . . . 133

Lipofectamine™ 2000 Transfection Reagent . . . . 132, 133

Lipofectamine™ 2000 CD Transfection Reagent . . . . 132

Lipofectamine™ LTX Transfection Reagent . . . . . . . 132

MMammalian Cells . . . . . . . . . . . . . . . . . . 177

293 Cells . . . . . . . . . . . . . . . . . . . . . 177

FreeStyle™ 293 Expression Medium . . . . . . . . . 177

CHO-S Cells . . . . . . . . . . . . . . . . . . . . 178

DG44 Cells . . . . . . . . . . . . . . . . . . . . 178

Primary Human Keratinocytes . . . . . . . . . . . 179

MarrowMAX™ . . . . . . . . . . . . . . . . . . . . 126

McCoy’s 5A Medium (Modified) . . . . . . . . . . . . .37

MCDB 131 Medium . . . . . . . . . . . . . . . . . .37

Media 199 . . . . . . . . . . . . . . . . . . . . . . .38

Medium 199 with Earle’s Salts . . . . . . . . . . . .38

Medium 199 (10X), liquid . . . . . . . . . . . . . . .38

Medium 199, powder . . . . . . . . . . . . . . . .38

Medium 199 with Hanks’ Salts . . . . . . . . . . . .38

Media Formulations . . . . . . . . . . . . . . . . . .50

Medium NCTC-109 . . . . . . . . . . . . . . . 42, 107

MEM Amino Acids Solution (50X), liquid . . . . . . . . 149

MEM Non-essential Amino Acids Solution 10 mM (100X), liquid . . . . . . . . . . . . . . . . . . . . . . . 149

MEM Vitamin Solution (100X), liquid . . . . . . . . . . 168

MesenPRO RS™ Medium . . . . . . . . . . . . . . . 118

Minimum Essential Media (MEM) . . . . . . . . . . . .39

Advanced Minimum Essential Medium (MEM) (1X),

liquid (with L-glutamine) . . . . . . . . . . . . . . .40

Minimum Essential Medium (MEM)

with Earle’s Salts (with L-glutamine) . . . . . . . . . .40


with Earle’s Salts (without L-glutamine) . . . . . . . .41


with Hanks’ Salts, liquid (with L-glutamine) . . . . . . .42

Modified Eagle Media . . . . . . . . . . . . . . . . .42

Mycophenolic Acid . . . . . . . . . . . . . . . . . 154

Mycoplasma Detection . . . . . . . . . . . . . . . 167

Yeast Extraction Solution . . . . . . . . . . . . . . 167

NN-2 Supplement . . . . . . . . . . . . . . . . . 115, 117

Natural Mouse Laminin . . . . . . . . . . . . . . . . 6

Neomycin Sulfate . . . . . . . . . . . . . . . . . . 154

Neurobasal™ Medium . . . . . . . . . . . . . . . . 117

Neurobiology Products . . . . . . . . . . . . . . . . 114

Neurobasal™ Media . . . . . . . . . . . . . . . . 114

B-27 Serum-free Supplements . . . . . . . . . . . 115

G-5 Supplement (100X), liquid . . . . . . . . . . . 115

N-2 Supplement (100X), liquid . . . . . . . . . . . 115

Brain-Derived Neurotrophic Factor (BDNF), Human, Recombinant . . . . . . . . . . . . . . . . . . 115

Nerve Growth Factor 2.5S (NGF 2.5S), Murine, Natural 116

Nerve Growth Factor 7S (NGF 7S), Murine, Natural . . 116

ViraPower™ Lentiviral Expression System . . . . . . 116

Newborn Calf Sera . . . . . . . . . . . . . . . . . . 191

Newborn Calf Serum . . . . . . . . . . . . . . . . 191

Newborn Calf Serum, heat-inactivated . . . . . . . 191

OOptiCHO™ Express Kit . . . . . . . . . . . . . . . . 180

OptiCHO™ Antibody Express Kit . . . . . . . . . . . . 181

Opti-MEM® I Reduced-Serum Media . . . . . . . . . . .43

Opti-MEM® I Reduced-Serum Medium (1X), liquid . . .43


(without phenol red) . . . . . . . . . . . . . . . . .43


(with GlutaMAX™-I) . . . . . . . . . . . . . . . . .43

Opti-MEM® I Reduced-Serum Medium, powder . . . .43

Lipofectamine™ 2000 Transfection Reagent . . . . . .43

OptiPRO™ SFM . . . . . . . . . . . . . . . . . . . 180

OpTmizer™ CTS™ T-Cell Expansion SFM . . . . . . . . . 3

Ordering and Technical Support . . . . . . . . . . . . v

PpcDNA™3.3-TOPO® TA Cloning® Kit . . . . . . . . . . 181

PD-Direct™ Bioprocess Services . . . . . . . . . . . . 209

Penicillin-Streptomycin, liquid . . . . . . . . . . 129, 154

Penicillin-Streptomycin-Glutamine (100X), liquid . . . . 154


Page Page Page

Alphabetical Index



Penicillin-Streptomycin-Neomycin (PSN) Antibiotic Mixture (100X), liquid . . . . . . . . . . 155

Performance Tests: Fetal Bovine Sera (FBS) . . . . . . . 197

PFHM-II Protein-free Hybridoma Medium (1X), liquid . . 107

Phenol Red–free Media at a glance . . . . . . . . . . .28

Phosphate-Buffered Saline (PBS) . . . . . . . . . . . 141

Phosphate-Buffered Saline (PBS) 7.2 (1X), liquid . . . 141




Phytohemagglutinin (M Form) (PHA), liquid . . . . . . 168

Pluronic® F-68, 10% (100X) . . . . . . . . . . . . . . 103

PLUS™ Reagent . . . . . . . . . . . . . . . . . . . 133

Polymyxin B Sulfate . . . . . . . . . . . . . . . . . 155

Porcine Serum . . . . . . . . . . . . . . . . . . . . 192

pOptiVEC™-TOPO® vector. . . . . . . . . . . . . . . 180

Prefilled Wave Cellbag . . . . . . . . . . . . . . . . 207

Presto Blue™ . . . . . . . . . . . . . . . . . . . . 170

Primary Human Keratinocytes . . . . . . . . . . . . 179

Protein A Agarose . . . . . . . . . . . . . . . . . . 108

Publication References . . . . . . . . . . . . . . . . .20

Puromycin . . . . . . . . . . . . . . . . . . . . . 151

RRabbit Serum . . . . . . . . . . . . . . . . . . . . 192

Rat proteins . . . . . . . . . . . . . . . . . . . . . .18

Recombinant human FGF-basic . . . . . . . . . . . .12

Recombinant Protein G (rProtein G) Agarose . . . . . . 108

Recovery™ Freezing Medium . . . . . . . . . . . . . .29

Reduced-serum Media at a glance . . . . . . . . . . .25

RPMI Media 1640 . . . . . . . . . . . . . . . . . . .44

RPMI Medium 1640 (1X), liquid . . . . . . . . . . . .45

RPMI Medium 1640, powder . . . . . . . . . . . . .45

RPMI Medium 1640 (1X), liquid (Folate-free) . . . . . 130

SSchneider’s Drosophila Medium . . . . . . . . . . . .46

Serum-Free, Protein-free, and Chemically Defined Media .85

Serum-free Media and Reagents . . . . . . . . . . . .86

Serum-free Media and Applications . . . . . . . . . . 220

Hybridoma Culture . . . . . . . . . . . . . . . . 220

Chinese Hamster Ovary (CHO) Cell Culture . . . . . 221

Human Embryonic Kidney (293) Cell Culture . . . . . 221

Mammalian Cell Culture for Virus Production . . . . 221

Insect Cell Culture . . . . . . . . . . . . . . . . . 222

Neuronal Cell Culture . . . . . . . . . . . . . . . 222

Blood and Bone Marrow Culture . . . . . . . . . . 223

Stem Cell Culture . . . . . . . . . . . . . . . . . 223

Other Mammalian Cell Culture . . . . . . . . . . . 223

Sf9 Cells . . . . . . . . . . . . . . . . . . . . . . . 175

Sf21 Cells . . . . . . . . . . . . . . . . . . . . . . 175

Sf-900™ III SFM . . . . . . . . . . . . . . . . . . . . .99

Sf-900™ II SFM . . . . . . . . . . . . . . . . . . . . .99

Sodium Bicarbonate Solution, 7.5% (w/v) . . . . . . . 130

Sodium Pyruvate Solution 100 mM (100X), liquid . . . . 168

Special Proccessing of Sera . . . . . . . . . . . . . . 200

Specialty FBS Selection Guide . . . . . . . . . . . . . 189

Specialty Media . . . . . . . . . . . . . . . . . . . .85

Specialty Media and Applications . . . . . . . . . . . 219

Specialty Reagents . . . . . . . . . . . . . . . . . .87

Stability of GlutaMAX™-I vs. L-Glutamine . . . . . . . . 226

Stak Pack™ Accessories . . . . . . . . . . . . . . . .49

Stem Cell Research Products . . . . . . . . . . . . . 117

B-27 Supplement . . . . . . . . . . . . . . . . . 117

N-2 Supplement . . . . . . . . . . . . . . . . . . 117

Neurobasal™ Medium . . . . . . . . . . . . . . . 117

Neurobasal™-A Medium . . . . . . . . . . . . . . 117

AIM V® Media (research grade) . . . . . . . . . . . 117

AIM V® Media (therapeutic grade) . . . . . . . . . . 118

MesenPRO RS™ Medium . . . . . . . . . . . . . . 118

StemPro® hESC SFM . . . . . . . . . . . . . . . . 119

StemPro®-34 SFM . . . . . . . . . . . . . . . . . 120

DMEM/F-12 . . . . . . . . . . . . . . . . . . . . 120

KnockOut™ Serum Replacement . . . . . . . . . . 120

KnockOut™ DMEM . . . . . . . . . . . . . . . . . 121

Geneticin® Selective Antibiotic . . . . . . . . . . . 122

Blasticidin S HCI . . . . . . . . . . . . . . . . . . 123

Hygromycin B . . . . . . . . . . . . . . . . . . . 123

GlutaMAX™-I Supplement . . . . . . . . . . . . . 123

Fetal Bovine Serum, ES Cell-Qualified . . . . . . . . 123

CO2-Independent Medium . . . . . . . . . . . . . 123

Basic Fibroblast Growth Factor (bFGF), Human,

Recombinant . . . . . . . . . . . . . . . . . . . 124

Insulin-like Growth Factor-I (IGF-I),

Human, Recombinant . . . . . . . . . . . . . . . 124

Platelet Derived Growth Factor BB (PDGF BB),


Streptavidin Agarose . . . . . . . . . . . . . . . . . 108

Streptomycin Sulfate . . . . . . . . . . . . . . . . . 155

Supply Centre by Life Technologiestm . . . . . . . . . 213

Swine proteins . . . . . . . . . . . . . . . . . . . .19

TThawing of Cryopreserved Cells . . . . . . . . . . . . 227

Thrombopoietin (TPO) . . . . . . . . . . . . . . . . .14

Transfection Reagents . . . . . . . . . . . . . . . . 132

Lipofectamine™ 2000 . . . . . . . . . . . . . . . 132

Lipofectamine™ LTX . . . . . . . . . . . . . . . . 132

FreeStyle™ MAX Reagent . . . . . . . . . . . . . . 132

293fectin™ Transfection Reagent . . . . . . . . . . 133

Lipofectamine™ RNAiMAX Transfection Reagent . . . 133

Cellfectin® Transfection Reagent . . . . . . . . . . 133

PLUS™ Reagent . . . . . . . . . . . . . . . . . . 133

Lipofectamine™ Transfection Reagent . . . . . . . . 133

Lipofectamine™ 2000 Transfection Reagent . . . . . 133

Troubleshooting: Cell Culture . . . . . . . . . . . . . 218

Trypan Blue Stain . . . . . . . . . . . . . . . . . . 168

U–ZUltralow IgG FBS . . . . . . . . . . . . . . . . . . . 190

Vascular-Endothelial Products . . . . . . . . . . . . . 124

Basic Fibroblast Growth Factor (bFGF),


Fibronectin, Human Plasma . . . . . . . . . . . . 125

Human Endothelial-SFM . . . . . . . . . . . . . . 124

Versene . . . . . . . . . . . . . . . . . . . . . . . 161

Viral Expression Products . . . . . . . . . . . . . . . .97

ViraPower™ Adenoviral Expression System . . . . . . .97

ViraPower™ Lentiviral Expression System . . . . . . 116

Water, distilled . . . . . . . . . . . . . . . . . . . . 130

Waymouth’s MB 752/1 Medium . . . . . . . . . . . .46

WFI for Cell Culture (Water for Injection) . . . . . . . 161

Williams’ Medium E . . . . . . . . . . . . . . . . . .46

Yeastolate Ultrafiltrate (50X), liquid . . . . . . . . . . 103

Zeocin™ . . . . . . . . . . . . . . . . . . . . . . . 155

Alphabetical Index

244


Notes:

gibco® cell culture catalogueAlphabetical Index



Notes:

Cat. no. Page Cat. no. Page Cat. no. Page Cat. no. Page Cat. no. Page

Important Licensing Information: The products in this catalog may covered by one or more Limited Use Label Licenses (see the Invitrogen website www.invitrogen.com). By use of these products, you accept the terms and conditions of all applicable Limited Use Label Licenses. All products are for research use only, and are not intended for human or animal diagnostics, therapeutic, commercial, or other clinical uses unless otherwise specifically indicated in the applicable product Limited Use Label License(s).

246

Catalogue Number Index

402-03D . . . . . . . . . . . . 3006-0096BA . . . . . . . . . 156006-0096BC . . . . . . . . . 1560070037BA . . . . . . . . 99, 2070070049BA . . . . . . . . . . 2070070049BB . . . . . . . . 93, 207087-0112BK . . . . . . . . . . 112087-0112BK . . . . . . . . . . 118087-0112BK . . . . . . . . . . . 3087-0112DK . . . . . . 3, 112, 11810010-001 . . . . . . . . . . 20610010-002 . . . . . . . . . . 20610010-015 . . . . . . . . . . 14110010-031 . . . . . . . . . . 14110010-056 . . . . . . . . . . 14110010-072 . . . . . . . . . . 14110082-139 . . . . . . . . . . 18710082-147 . . . . . . . . . . 18710092-013 . . . . . . . . . . 12810099-133 . . . . . . . . . . 18810099-141 . . . . . . . . . . 18810100-139 . . . . . . . . . . 18810100-147 . . . . . . . . . . 18810106-151 . . . . . . . . . . 18810106-169 . . . . . . . . . . 18810108-157 . . . . . . . . . . 18810108-165 . . . . . . . . . . 18810109-155 . . . . . . . . . . 18910109-163 . . . . . . . . . . 18910131-019 . . . . . . . . . . 12210131-027 . . . . . . . . . . 12210131-027 . . . . . . . . . . 15210131-027 . . . . . . . . . . 18110131-035 . . . . . . . . . . 15210177-012 . . . . . . . . . . 15110265-015 . . . . . . . . . . .4910270-098 . . . . . . . . . . 18810270-106 . . . . . . . . . . 18810362-010 . . . . . . . . . . 13310362-100 . . . . . . . . . . 10310362-125 . . . . . . . . . . 10310370-047 . . . . . . . . . . .4110370-070 . . . . . . . . . . .4110371-029 . . . . . . . . . . 19110372-019 . . . . . . . . . . .3710373-017 . . . . . . . . . . .3610378-016 . . . . . . . . . . 15410439-016 . . . . . . . . . . 12310439-024 . . . . . . . . . . 12310486-025 . . . . . . . . . . 10010500-056 . . . . . . . . . . 18810500-064 . . . . . . . . . . 18810564-037 . . . . . . . . . . 20610564-045 . . . . . . . . . . 20610566-032 . . . . . . . . . . 20610566-040 . . . . . . . . . . 20610569-069 . . . . . . . . . . 20610569-077 . . . . . . . . . . 20610575-090 . . . . . . . . . . 12810576-015 . . . . . . . . . . 12810576-015 . . . . . . . . . . 16810582-013 . . . . . . . . . . 12810639-011 . . . . . . . . . . 11310639-011 . . . . . . . . . . 12010687-010 . . . . . . . . . . 12310687-010 . . . . . . . . . . 15310743-001 . . . . . . . . . . 20610743-002 . . . . . . . . . . 206

10744-019 . . . . . . . . . . 11010828-028 . . . . . . . . . . 12010829-018 . . . . . . . . . . 12110831-014 . . . . . . . . . . 17410833-010 . . . . . . . . . . .4910833-028 . . . . . . . . . . .4910833-036 . . . . . . . . . . .4910888-022 . . . . . . . . . . 11410888-022 . . . . . . . . . . 11710889-038 . . . . . . . . . . 11510889-038 . . . . . . . . . . 11710889-038 . . . . . . . . . . 16410902-088 . . . . . . . . . . .9910902-096 . . . . . . . . . . .9910902-104 . . . . . . . . . . .9910902-161 . . . . . . . . 99, 20610902-179 . . . . . . . . . . 20610902-187 . . . . . . . . 99, 20610908-010 . . . . . . . . . . 11510967-032 . . . . . . . . . . 10011003-019 . . . . . . . . . . .4911020-021 . . . . . . . . . . 16311020-039 . . . . . . . . . . 16311021-029 . . . . . . . . . . 16311021-037 . . . . . . . . . . 16311021-045 . . . . . . . . . . 16311039-021 . . . . . . . . . . .3311039-047 . . . . . . . . . . .3311058-021 . . . . . . . . . . .4311058-021 . . . . . . . . . . .9511067-030 . . . . . . . . . . 10711067-030 . . . . . . . . . . 16611105-012 . . . . . . . . . . 16511107-018 . . . . . . . . . . 16511107-047 . . . . . . . . . . 16511108-016 . . . . . . . . . . 16511111-044 . . . . . . . . . . 12511120-037 . . . . . . . . . . 16811126-034 . . . . . . . . . . .2911130-036 . . . . . . . . . . 14911130-077 . . . . . . . . . . 14911140-035 . . . . . . . . . . 14911140-068 . . . . . . . . . . 14911269-016 . . . . . . . . . . 12711279-023 . . . . . . . . . . 10611300-027 . . . . . . . . . . .3511330-099 . . . . . . . . . . 20611330-107 . . . . . . . . 33, 20611344-041 . . . . . . . . . . 15711360-039 . . . . . . . . . . 16811360-088 . . . . . . . . . . 16811405-057 . . . . . . . . . . .3611405-081 . . . . . . . . . . 10011415-049 . . . . . . . . . . .3711415-056 . . . . . . . . . . .3711496-015 . . . . . . . . . . 17511497-013 . . . . . . . . . . 17611514-015 . . . . . . . . . . 13311593-027 . . . . . . . . . . 15011595-030 . . . . . . . . . . 10011595-030 . . . . . . . . . . .3511605-045 . . . . . . . . . . 10011605-045 . . . . . . . . . . .3511605-086 . . . . . . . . . . 10011605-086 . . . . . . . . . . .3511619-012 . . . . . . . . . . 17811625-019 . . . . . . . . . . 17711631-017 . . . . . . . . . . 177

11667-037 . . . . . . . . . . 10011667-037 . . . . . . . . . . .3511668-019 . . . . . . . . . . 13211668-019 . . . . . . . . . . 13311668-019 . . . . . . . . . . .4311668-027 . . . . . . . . . . 13211668-027 . . . . . . . . . . 13311668-027 . . . . . . . . . . .4311668-500 . . . . . . . . . . 13211668-500 . . . . . . . . . . 13311681-020 . . . . . . . . . . .9511686-029 . . . . . . . . . . .9311686-029 . . . . . . . . . . .9711765-047 . . . . . . . . . . .3411800-042 . . . . . . . . . . 16511800-042 . . . . . . . . . . 16711805-017 . . . . . . . . . . 15011811-023 . . . . . . . . . . 12211811-023 . . . . . . . . . . 15311811-031 . . . . . . . . . . 12211811-031 . . . . . . . . . . 15311811-064 . . . . . . . . . . 12211811-064 . . . . . . . . . . 15311811-098 . . . . . . . . . . 12211811-098 . . . . . . . . . . 15311814-019 . . . . . . . . . . 15411815-024 . . . . . . . . . . 15311815-032 . . . . . . . . . . 15311835-063 . . . . . . . . . . .4511860-038 . . . . . . . . . . 15511875-168 . . . . . . . . . . 20611875-176 . . . . . . . . . . 20611879-020 . . . . . . . . . . .4511880-028 . . . . . . . . . . 11811880-028 . . . . . . . . . . .3211900-073 . . . . . . . . . . .4011905-031 . . . . . . . . . . 10311905-031 . . . . . . . . . . 16311905-031 . . . . . . . . . . 16611905-031 . . . . . . . . . . 16711913-019 . . . . . . . . . . .9311913-019 . . . . . . . . . . .9611960-044 . . . . . . . . . . .3011960-085 . . . . . . . . . . .3011965-167 . . . . . . . . . . 20611965-175 . . . . . . . . . . 20611966-025 . . . . . . . . . . .3211971-025 . . . . . . . . . . .3011995-115 . . . . . . . . . . 20611995-123 . . . . . . . . . . 20612000-063 . . . . . . . . . . .4012002-010 . . . . . . . . . . .4912040-077 . . . . . . . . . . 10712045-076 . . . . . . . . . . 10612045-084 . . . . . . . . . . 10612055-083 . . . . . . . . . . 11212055-083 . . . . . . . . . . 11712055-091 . . . . . . . . . . 11212055-091 . . . . . . . . . . 11712065-074 . . . . . . . . . . 11312102 . . . . . . . . . . . . . 312260-014 . . . . . . . . . . 12612309-019 . . . . . . . . . . .9412309-050 . . . . . . . . . . 18012309-050 . . . . . . . . . . 18112309-050 . . . . . . . . . . .9212332-011 . . . . . . . . . . 17912338-001 . . . . . . . . . . 206

12338-002 . . . . . . . . . . 20612338-018 . . . . . . . . . . .9312338-018 . . . . . . . . . . .9412338-026 . . . . . . . . . . 17712338-026 . . . . . . . . . . .9312347-019 . . . . . . . . . . 13312347-019 . . . . . . . . . . .9412347-500 . . . . . . . . . . 13312347-750 . . . . . . . . . . 13312348-017 . . . . . . . . . . 11412349-015 . . . . . . . . . . 11412372-017 . . . . . . . . . . 10612372-025 . . . . . . . . . . 10612430-112 . . . . . . . . . . 20612430-120 . . . . . . . . . . 20612440-046 . . . . . . . . . . .3612491-015 . . . . . . . . . . .3012491-023 . . . . . . . . . . .3012492-013 . . . . . . . . . . .4012492-021 . . . . . . . . . . .4012529-012 . . . . . . . . . . .9312529-012 . . . . . . . . . . .9712529-020 . . . . . . . . . . .9312529-020 . . . . . . . . . . .9712531-018 . . . . . .106, 163, 16612556-015 . . . . . . . . . . 12712556-023 . . . . . . . . . . 12712557-013 . . . . . . . . . . 12712557-021 . . . . . . . . . . 12712558-011 . . . . . . . . . . 12712559-019 . . . . . . . . . . .9512559-027 . . . . . . . . . . .9512563-011 . . . . . . . . . . 15912563-011 . . . . . . . . . . .8712563-029 . . . . . . . . . . 15912563-029 . . . . . . . . . . .8712566-014 . . . . . . . . . . 13212568-010 . . . . . . . . . . 11012582-011 . . . . . . . . . . .9612585-014 . . . . . . . . . . 16412587-010 . . . . . . . . . . 11512587-010 . . . . . . . . . . 11712587-010 . . . . . . . . . . 16412591-038 . . . . . . . . . . .2812604-013 . . . . . . . . . . 11912604-013 . . . . . . . . . . 16012604-021 . . . . . . . . . . 11812604-021 . . . . . . . . . . 16012604-039 . . . . . . . . . . 16012604-054 . . . . . . . . 160, 20612605-010 . . . . . . . . . . 16012605-028 . . . . . . . . . . 16012605-036 . . . . . . . . . . 16012605-093 . . . . . . . . 160, 20612610-010 . . . . . . . . . . 18012610-010 . . . . . . . . . . 18112610-010 . . . . . . . . . . .9212618-012 . . . . . . . . . . . 412618-013 . . . . . . . . . . . 412633-012 . . . . . . . . . . .4512633-020 . . . . . . . . . . .4512634-010 . . . . . . . . . . .3312634-028 . . . . . . . . . . .3312648-010 . . . . . . . . . . .2912658-001 . . . . . . . . . . 20612658-019 . . . . . . . . . . .9912658-027 . . . . . . . . . . .9912658-035 . . . . . . . . 99, 206

12659-017 . . . . . . . . . . 17512661-013 . . . . . . . . . . .9612662-011 . . . . . . . . . . 19012662-029 . . . . . . . . . . 19012676-011 . . . . . . . . . . 18912676-029 . . . . . . . . . . 18912677-019 . . . . . . . . . . 10912677-027 . . . . . . . . . . 10912678-017 . . . . . . . . . . 10912681-011 . . . . . . . . . . 18012681-011 . . . . . . . . . . 18112681-011 . . . . . . . . . . .8912681-011 . . . . . . . . . . .9212681-029 . . . . . . . . . . .8912682-019 . . . . . . . . . . 17512684-015 . . . . . . . . . . .8812684-031 . . . . . . . . . . .8812684015 . . . . . . . . . . . . 612684023 . . . . . . . . . . . . 612684031 . . . . . . . . . . . . 612684049 . . . . . . . . . . . . 612744-017 . . . . . . . . . . 18012744-017 . . . . . . . . . . 18112744-017 . . . . . . . . . . .9212746-012 . . . . . . . . . . 11812752-010 . . . . . . . . . . 11412753-018 . . . . . . . . . . 11412755-013 . . . . . . . . . . 11412760-013 . . . . . . . . . . .8812760-021 . . . . . . . . . . 11912760-021 . . . . . . . . . . .8812760013 . . . . . . . . . . . . 512760021 . . . . . . . . . . . . 512800-116 . . . . . . . . . . .3013018-015 . . . . . . . . . . .4513018-031 . . . . . . . . . . .4513150-016 . . . . . . . . . . 15813151-014 . . . . . . . . . . 15813256-029 . . . . . . . . . . 12513257-019 . . . . . . . . . . 11613290-010 . . . . . . . . . . 11613778-075 . . . . . . . . . . 13313778-150 . . . . . . . . . . 13314025-050 . . . . . . . . . . 14014025-076 . . . . . . . . . . 14014025-100 . . . . . . . . . . 14014025-126 . . . . . . . . . . 14014040-083 . . . . . . . . . . 13814040-091 . . . . . . . . . . 13814040-117 . . . . . . . . . . 13814040-174 . . . . . . . . . . 13814040-224 . . . . . . . . . . 13814065-049 . . . . . . . . . . 14014080-048 . . . . . . . . . . 13914155-048 . . . . . . . . . . 13914170-070 . . . . . . . . . . 14014170-088 . . . . . . . . . . 14014170-138 . . . . . . . . . . 14014170-195 . . . . . . . . 140, 20614170-203 . . . . . . . . 140, 20614175-053 . . . . . . . . . . 14014175-079 . . . . . . . . . . 14014175-129 . . . . . . . . . . 14014175-145 . . . . . . . . . . 14014185-045 . . . . . . . . . . 14014190-094 . . . . . . . . . . 11814190-094 . . . . . . . . . . 11914190-094 . . . . . . . . . . 138






14190-136 . . . . . . . . . . 13814190-169 . . . . . . . . . . 13814190-326 . . . . . . . . . . 13814190-342 . . . . . . . . 138, 20614190-359 . . . . . . . . 138, 20614190-367 . . . . . . . . 138, 20614200-067 . . . . . . . . . . 13914200-083 . . . . . . . . . . 13914287-072 . . . . . . . . . . 13814287-080 . . . . . . . . . . 13815023-021 . . . . . . . . . . 16815040-033 . . . . . . . . . . 16115070-063 . . . . . . . . . . 12915070-063 . . . . . . . . . . 15415090-046 . . . . . . . . . . 16015140-122 . . . . . . . . . . 12915140-122 . . . . . . . . . . 15415140-130 . . . . . . . . . . 15415140-148 . . . . . . . . . . 12915140-148 . . . . . . . . . . 15415140-163 . . . . . . . . . . 12915160-047 . . . . . . . . . . 15315160-070 . . . . . . . . . . 15315210-040 . . . . . . . . . . 12715212-040 . . . . . . . . . . 12815230-001 . . . . . . . . . . 16115230-071 . . . . . . . . . . 13015230-071 . . . . . . . . . . 16115230-089 . . . . . . . . . . 16115230-097 . . . . . . . . . . 16115230-147 . . . . . . . . . . 13015230-162 . . . . . . . . . . 13015230-196 . . . . . . . . . . 13015230-196 . . . . . . . . . . 16115230-204 . . . . . . . . . . 16115230-253 . . . . . . . . 161, 20615230-261 . . . . . . . . 161, 20615230-279 . . . . . . . . . . 20615230-788 . . . . . . . . . . 13015240-062 . . . . . . . . . . 12915240-062 . . . . . . . . . . 15015240-096 . . . . . . . . . . 12915240-096 . . . . . . . . . . 15015240-112 . . . . . . . . . . 12915250-061 . . . . . . . . . . 16815260-037 . . . . . . . . . . 16315290-018 . . . . . . . . . . 12915290-026 . . . . . . . . . . 12915338-100 . . . . . . . . . . 13215338-500 . . . . . . . . . . 13215400-054 . . . . . . . . . . 16015630-049 . . . . . . . . . . 15715630-056 . . . . . . . . . . 15715630-122 . . . . . . . . . . 15715640-055 . . . . . . . . . . 15515710-049 . . . . . . . . . . 11815710-049 . . . . . . . . . . 15315710-064 . . . . . . . . . . 12915710-072 . . . . . . . . . . 12915710-080 . . . . . . . . . . 15315750-045 . . . . . . . . . . 15315750-060 . . . . . . . . . . 12915750-078 . . . . . . . . . . 12915750-102 . . . . . . . . . . 15315918-014 . . . . . . . . . . 10815920-010 . . . . . . . . . . 10816000-036 . . . . . . . . . . 18716000-044 . . . . . . . . . . 187

16010-159 . . . . . . . . . . 19116010-167 . . . . . . . . . . 19116030-074 . . . . . . . . . . 19116050-122 . . . . . . . . . . 19216050-130 . . . . . . . . . . 19216070-096 . . . . . . . . . . 19216110-082 . . . . . . . . . . 19216120-099 . . . . . . . . . . 19216120-107 . . . . . . . . . . 19216140-063 . . . . . . . . . . 18816140-071 . . . . . . . . . . 18816141-061 . . . . . . . . . . 12316141-061 . . . . . . . . . . 18916141-079 . . . . . . . . . . 12316141-079 . . . . . . . . . . 18916170-078 . . . . . . . . . . 19116170-086 . . . . . . . . . . 19116210-072 . . . . . . . . . . 19216210-096 . . . . . . . . . . 19216250-078 . . . . . . . . . . 19016250-086 . . . . . . . . . . 19016447-100 . . . . . . . . . . 13216447-100 . . . . . . . . . . 18016447-100 . . . . . . . . . . 18116447-100 . . . . . . . . . . .9216447-500 . . . . . . . . . . 13216447-750 . . . . . . . . . . 13217001-074 . . . . . . . . . . 12717001-082 . . . . . . . . . . 12717005-042 . . . . . . . . . . 11017018-029 . . . . . . . . . . 13017018-029 . . . . . . . . . . 15817075-029 . . . . . . . . . . 16117100-017 . . . . . . . . . . 15817101-015 . . . . . . . . . . 15817104-019 . . . . . . . . . . 11917104-019 . . . . . . . . . . 15817105-041 . . . . . . . . . . 15817502-048 . . . . . . . . . . 11517502-048 . . . . . . . . . . 11717502-048 . . . . . . . . . . 16517503-012 . . . . . . . . . . 11517503-012 . . . . . . . . . . 16417504-044 . . . . . . . . . . 11517504-044 . . . . . . . . . . 16417701-038 . . . . . . . . . . 11317703-034 . . . . . . . . . . 11317704-024 . . . . . . . . . . 11317705-021 . . . . . . . . . . 11318045-054 . . . . . . . . . . 12318045-054 . . . . . . . . . . .2918045-070 . . . . . . . . . . 12318180-059 . . . . . . . . . . 16718200-048 . . . . . . . . . . 10318200-048 . . . . . . . . . . 16718300-012 . . . . . . . . . . 10318300-012 . . . . . . . . . . 16718324-012 . . . . . . . . . . 13318324-020 . . . . . . . . . . 13318324-111 . . . . . . . . . . 13320012-019 . . . . . . . . . . 14120012-043 . . . . . . . . . . 14120012-068 . . . . . . . . . . 14121012-026 . . . . . . . . . . .9921013-024 . . . . . . . . . . .3021013-024 . . . . . . . . . . .3221041-025 . . . . . . . . . . .3321051-040 . . . . . . . . . . 149

21056-023 . . . . . . . . . . .3621060-017 . . . . . . . . . . 10721060-017 . . . . . . . . . . 16621063-029 . . . . . . . . . . .3021083-027 . . . . . . . . . . .3721090-022 . . . . . . . . . . .4121090-055 . . . . . . . . . . .4121103-049 . . . . . . . . . . 11421103-049 . . . . . . . . . . 11721127-022 . . . . . . . . . . .3421180-021 . . . . . . . . . . .3821300-017 . . . . . . . . . . 13921300-058 . . . . . . . . . . 13921340-054 . . . . . . . . . . 10721340-054 . . . . . . . . . . .4221430-020 . . . . . . . . . . .4121530-027 . . . . . . . . . . .2921540-026 . . . . . . . . . . .2921575-022 . . . . . . . . . . .4221600-010 . . . . . . . . . . 13921600-044 . . . . . . . . . . 13921600-069 . . . . . . . . . . 13921700-018 . . . . . . . . . . .3421700-026 . . . . . . . . . . .3421710-025 . . . . . . . . . . .3521720-024 . . . . . . . . . . 10021720-024 . . . . . . . . . . .4621765-029 . . . . . . . . . . .3421765-037 . . . . . . . . . . .3421810-031 . . . . . . . . . . 15421850-029 . . . . . . . . . . 15521875-034 . . . . . . . . . . .4521875-042 . . . . . . . . . . .4521875-091 . . . . . . . . . . .4521875-158 . . . . . . . . . . .4521885-025 . . . . . . . . . . .3221885-108 . . . . . . . . . . .3221969-035 . . . . . . . . . . .3021980-032 . . . . . . . . . . .3621980-065 . . . . . . . . . . .3621985-023 . . . . . . . . . . 16822320-022 . . . . . . . . . . .3222330-021 . . . . . . . . . . .3722340-020 . . . . . . . . . . .3822350-029 . . . . . . . . . . .3822390-025 . . . . . . . . . . .3422400-147 . . . . . . . . . . 20622400-154 . . . . . . . . . . 20622551-022 . . . . . . . . . . .4622561-021 . . . . . . . . . . .4022561-054 . . . . . . . . . . .4022571-020 . . . . . . . . . . .4022571-038 . . . . . . . . . . .4022600-050 . . . . . . . . . . .9522600-134 . . . . . . . . . . .4322600-134 . . . . . . . . . . .9523017-015 . . . . . . . . . . 16223017-015 . . . . . . . . . . . 623181-010 . . . . . . . . . . 11924020-091 . . . . . . . . . . 14024040-032 . . . . . . . . . . 10324040-032 . . . . . . . . . . 16724040-032 . . . . . . . . . . 18024040-032 . . . . . . . . . . 18124040-032 . . . . . . . . . . .9225030-024 . . . . . . . . . . 11825030-032 . . . . . . . . . . 14925030-081 . . . . . . . . . . 130

25030-081 . . . . . . . . . . 18025030-081 . . . . . . . . . . 18125030-081 . . . . . . . . . . .9225030-123 . . . . . . . . . . 13025030-123 . . . . . . . . . . 14925030-149 . . . . . . . . . . 13025050-014 . . . . . . . . . . 16025050-030 . . . . . . . . . . 16025080-060 . . . . . . . . . . 13025080-060 . . . . . . . . . . 15725080-102 . . . . . . . . . . 13025200-056 . . . . . . . . . . 16025200-072 . . . . . . . . . . 16025200-114 . . . . . . . . . . 16025300-054 . . . . . . . . . . 16025300-062 . . . . . . . . . . 16025300-096 . . . . . . . . . . 16025300-104 . . . . . . . . . . 16026010-066 . . . . . . . . . . 19126010-074 . . . . . . . . . . 19126050-070 . . . . . . . . . . 19226050-088 . . . . . . . . . . 19226140-079 . . . . . . . . . . 18826140-087 . . . . . . . . . . 18826170-035 . . . . . . . . . . 19126170-043 . . . . . . . . . . 19126250-084 . . . . . . . . . . 19226400-036 . . . . . . . . . . 18926400-044 . . . . . . . . . . 18926600-023 . . . . . . . . . . .3726600-080 . . . . . . . . . . .3727016-021 . . . . . . . . . . 13027016-021 . . . . . . . . . . .4527250-018 . . . . . . . . . . 16131035-025 . . . . . . . . . . 11231035-025 . . . . . . . . . . 11731053-028 . . . . . . . . . . .3031095-029 . . . . . . . . . . .4031095-052 . . . . . . . . . . .4031100-019 . . . . . . . . . . .3831100-027 . . . . . . . . . . .3831150-022 . . . . . . . . . . .3831150-030 . . . . . . . . . . .3831220-023 . . . . . . . . . . .4631330-038 . . . . . . . . . . .3331330-095 . . . . . . . . . . .3331331-028 . . . . . . . . . . 12031331-028 . . . . . . . . . . .3331331-093 . . . . . . . . . . 12031331-093 . . . . . . . . . . .3331350-010 . . . . . . . . . . 11931550-023 . . . . . . . . . . .3431600-083 . . . . . . . . . . .3231600-091 . . . . . . . . . . .3231765-027 . . . . . . . . . . .3431765-068 . . . . . . . . . . .3431870-025 . . . . . . . . . . .4531870-074 . . . . . . . . . . .4531885-023 . . . . . . . . . . .3231885-049 . . . . . . . . . . .3231966-021 . . . . . . . . . . .3131966-047 . . . . . . . . . . .3131980-022 . . . . . . . . . . .3631980-048 . . . . . . . . . . .3631985-047 . . . . . . . . . . .4331985-047 . . . . . . . . . . .9431985-054 . . . . . . . . . . .4331985-054 . . . . . . . . . . .94

31985-062 . . . . . . . . . . .4331985-062 . . . . . . . . . . .9432360-026 . . . . . . . . . . .4132430-027 . . . . . . . . . . .3132430-100 . . . . . . . . . . .3132561-029 . . . . . . . . . . .4132561-094 . . . . . . . . . . .4132571-028 . . . . . . . . . . .4132571-093 . . . . . . . . . . .4133016-015 . . . . . . . . . . 12533016-015 . . . . . . . . . . 16235050-038 . . . . . . . . . . 12335050-038 . . . . . . . . . . 14935050-061 . . . . . . . . . . 13035050-079 . . . . . . . . . . 13035050-087 . . . . . . . . . . 12335050-087 . . . . . . . . . . 14937010-022 . . . . . . . . . . 11039-8401 . . . . . . . . . . . 118402-03D . . . . . . . . . . . . 341-1000 . . . . . . . . . . . 11941-1100 . . . . . . . . . . . 11941-1200 . . . . . . . . . . . 11941-4000 . . . . . . . . . . . 11941-4400 . . . . . . . . . . . 11941010-026 . . . . . . . . . . .2841061-029 . . . . . . . . . . .4041090-028 . . . . . . . . . . .4141090-093 . . . . . . . . . . .4141300-021 . . . . . . . . . . .3741300-070 . . . . . . . . . . .3741400-045 . . . . . . . . . . 16441500-018 . . . . . . . . . . .4041500-083 . . . . . . . . . . .4041965-039 . . . . . . . . . . .3041965-047 . . . . . . . . . . .3041965-062 . . . . . . . . . . .3041965-120 . . . . . . . . . . .3041966-029 . . . . . . . . . . .3041966-052 . . . . . . . . . . .3042200-014 . . . . . . . . . . .3642200-030 . . . . . . . . . . .3642360-024 . . . . . . . . . . .4142360-081 . . . . . . . . . . .4142430-025 . . . . . . . . . . .3042430-082 . . . . . . . . . . .3051200-046 . . . . . . . . . . .4151300-044 . . . . . . . . . . 16451500-056 . . . . . . . . . . 16551800-019 . . . . . . . . . . .4551800-035 . . . . . . . . . . .4551800-043 . . . . . . . . . . .4551985-026 . . . . . . . . . . .4351985-026 . . . . . . . . . . .9451985-042 . . . . . . . . . . .4351985-042 . . . . . . . . . . .9452100-039 . . . . . . . . . . .3052400-017 . . . . . . . . . . .4552400-025 . . . . . . . . . . .4552400-041 . . . . . . . . . . .4561100-087 . . . . . . . . . . .4061100-103 . . . . . . . . . . .4061870-010 . . . . . . . . . . .4561870-044 . . . . . . . . . . .4561870-143 . . . . . . . . . . 20661870-150 . . . . . . . . . . 20661965-026 . . . . . . . . . . .3161965-059 . . . . . . . . . . .31


Catalog Number Index


248

70011-036 . . . . . . . . . . 14170011-051 . . . . . . . . . . 14170013-016 . . . . . . . . . . 14170013-065 . . . . . . . . . . 14172400-021 . . . . . . . . . . .4572400-054 . . . . . . . . . . .4572400-146 . . . . . . . . . . 20672400-153 . . . . . . . . . . 206

A–BA1000701 . . . . . . . . . . 119A10142-01 . . . . . . . . . . . 4A1014201 . . . . . . . . . . . 4A10234-01 . . . . . . . . . . .91A10240-01 . . . . . . . . . . .91A10241-01 . . . . . . . . . . .91A1036901 . . . . . . . . . . 156A1036902 . . . . . . . . . . 156A1048001 . . . . . . . . . . . 5A1048002 . . . . . . . . . . . 5A1048301 . . . . . . . . . . . 5A1048301 . . . . . . . . . . .88A10485-01 . . . . . . . . . . . 3A1049201 . . . . . . . . . . 149A1064401 . . . . . . . . . . . 5A1091501 . . . . . . . . . . . 6A1098201 . . . . . . . . . . . 6A1098202 . . . . . . . . . . . 6A1099201 . . . . . . . . . . . 4A1099202 . . . . . . . . . . . 4A1099901 . . . . . . . . . . .92A1100-001 . . . . . . . . . . .91A1113-802 . . . . . . . . . . 151A1113-803 . . . . . . . . . . 151A1113-902 . . . . . . . . . . 151A1113-903 . . . . . . . . . . 151A1134001 . . . . . . . . . . . 6A1134301 . . . . . . . . . . . 5A1142801 . . . . . . . . . . . 5A1142802 . . . . . . . . . . . 5A1142803 . . . . . . . . . . . 5A12856-01 . . . . . . . . . . 3, 4A12858-01 . . . . . . . . . . 3, 4A12859-01 . . . . . . . . . . . 4A12860-01 . . . . . . . . . . . 4A12861-01 . . . . . . . . . . . 4A1287-301 . . . . . . . . . . 161A1287-302 . . . . . . . . . . 161A1287-303 . . . . . . . . . . 161A1287-304 . . . . . . . . . . 161A13261 . . . . . . . . . . . . 171A13262 . . . . . . . . . . . . 171B855-02 . . . . . . . . . . . 174

C–MCTP0021 . . . . . . . . . . . . 3CTP0023 . . . . . . . . . . . . 3CTP0041 . . . . . . . . . . . . 3CTP0043 . . . . . . . . . . . . 3CTP0061 . . . . . . . . . . . . 4CTP0063 . . . . . . . . . . . . 4CTP0071 . . . . . . . . . . . . 3CTP0073 . . . . . . . . . . . . 3CTP0261 . . . . . . . . . . . . 4CTP0263 . . . . . . . . . . . . 4CTP2011 . . . . . . . . . . . . 3CTP2013 . . . . . . . . . . . . 3

CTP2111 . . . . . . . . . . . . 4CTP2113 . . . . . . . . . . . . 4CTP3011 . . . . . . . . . . . . 3CTP3013 . . . . . . . . . . . . 3CTP9211 . . . . . . . . . . . . 4CTP9213 . . . . . . . . . . . . 4CTP9411 . . . . . . . . . . . . 4CTP9413 . . . . . . . . . . . . 4DGCP10 . . . . . . . . . . . 183DGCP20 . . . . . . . . . . . 183DGCS10 . . . . . . . . . . . 183DGCS20 . . . . . . . . . . . 183HMCPIL . . . . . . . . . . . 183HMCPIS . . . . . . . . . . . 183HMCPML . . . . . . . . . . . 183HMCPMS . . . . . . . . . . . 183HMCPTL . . . . . . . . . . . 183HMCPTS . . . . . . . . . . . 183HMCS1L . . . . . . . . . . . 183HMCS1S . . . . . . . . . . . 183HMCS2L . . . . . . . . . . . 183HMCS2S . . . . . . . . . . . 183HMCS3L . . . . . . . . . . . 183HMCS3S . . . . . . . . . . . 183HMCSTL . . . . . . . . . . . 183HMCSTS . . . . . . . . . . . 183K4930-00 . . . . . . . . . . . .97K4945-00 . . . . . . . . . . . .97K4950-00 . . . . . . . . . . . 116K4960-00 . . . . . . . . . . . 116K4965-00 . . . . . . . . . . . 116K4980-00 . . . . . . . . . . . 116K4990-00 . . . . . . . . . . . 116K5910-00 . . . . . . . . . . . 116K8300-01 . . . . . . . . . . . 181K9000-01 . . . . . . . . . . . .94K9000-20 . . . . . . . . . . . .89MKCP10 . . . . . . . . . . . 183MKCP20 . . . . . . . . . . . 183MKCS10 . . . . . . . . . . . 183MKCS20 . . . . . . . . . . . 183MSCP10 . . . . . . . . . . . 183MSCP20 . . . . . . . . . . . 183MSCS10 . . . . . . . . . . . 183MSCS20 . . . . . . . . . . . 183

PPHB0044 . . . . . . . . . . . .15PHB0064 . . . . . . . . . . . .15PHC0011 . . . . . . . . . . . .16PHC0013 . . . . . . . . . . . .16PHC0015 . . . . . . . . . . . .16PHC0017 . . . . . . . . . . . .16PHC0021 . . . . . . . . . . . .16PHC0023 . . . . . . . . . . . .16PHC0026 . . . . . . . . . . . .16PHC0027 . . . . . . . . . . . .16PHC0031 . . . . . . . . . . . .16PHC0033 . . . . . . . . . . . .16PHC0034 . . . . . . . . . . . .16PHC0035 . . . . . . . . . . . .16PHC0041 . . . . . . . . . . . .16PHC0043 . . . . . . . . . . . .16PHC0044 . . . . . . . . . . . .16PHC0045 . . . . . . . . . . . .16PHC0052 . . . . . . . . . . . .15PHC0055 . . . . . . . . . . . .16

PHC0061 . . . . . . . . . . . .16PHC0063 . . . . . . . . . . . .16PHC0064 . . . . . . . . . . . .16PHC0065 . . . . . . . . . . . .16PHC0066 . . . . . . . . . . . .16PHC0071 . . . . . . . . . . . .16PHC0073 . . . . . . . . . . . .16PHC0075 . . . . . . . . . . . .16PHC0076 . . . . . . . . . . . .16PHC0084 . . . . . . . . . . . .16PHC0101 . . . . . . . . . . . .16PHC0103 . . . . . . . . . . . .16PHC0104 . . . . . . . . . . . .16PHC0105 . . . . . . . . . . . .16PHC0115 . . . . . . . . . . . .16PHC0131 . . . . . . . . . . . .16PHC0134 . . . . . . . . . . . .16PHC0135 . . . . . . . . . . . .16PHC0174 . . . . . . . . . . . .16PHC0186 . . . . . . . . . . . .16PHC0191 . . . . . . . . . . . .16PHC0194 . . . . . . . . . . . .16PHC0201 . . . . . . . . . . . .16PHC0204 . . . . . . . . . . . .16PHC0205 . . . . . . . . . . . .16PHC0211 . . . . . . . . . . . .16PHC0213 . . . . . . . . . . . .16PHC0214 . . . . . . . . . . . .16PHC0215 . . . . . . . . . . . .16PHC0811 . . . . . . . . . . . .16PHC0813 . . . . . . . . . . . .16PHC0814 . . . . . . . . . . . .16PHC0815 . . . . . . . . . . . .16PHC0816 . . . . . . . . . . . .16PHC0881 . . . . . . . . . . . .16PHC0883 . . . . . . . . . . . .16PHC0884 . . . . . . . . . . . .16PHC0885 . . . . . . . . . . . .16PHC1011 . . . . . . . . . . . .16PHC1014 . . . . . . . . . . . .16PHC1015 . . . . . . . . . . . .16PHC1031L . . . . . . . . . . .17PHC1033L . . . . . . . . . . .17PHC1034Z . . . . . . . . . . .17PHC1051 . . . . . . . . . . . .17PHC1053 . . . . . . . . . . . .17PHC1054 . . . . . . . . . . . .17PHC1055 . . . . . . . . . . . .17PHC1061 . . . . . . . . . . . .16PHC1063 . . . . . . . . . . . .16PHC1065 . . . . . . . . . . . .16PHC1066 . . . . . . . . . . . .16PHC1074 . . . . . . . . . . . .16PHC1081 . . . . . . . . . . . .16PHC1083 . . . . . . . . . . . .16PHC1084 . . . . . . . . . . . .16PHC1085 . . . . . . . . . . . .16PHC1094 . . . . . . . . . . . .17PHC1095 . . . . . . . . . . . .17PHC1101 . . . . . . . . . . . .17PHC1104 . . . . . . . . . . . .17PHC1111 . . . . . . . . . . . .16PHC1114 . . . . . . . . . . . .16PHC1115 . . . . . . . . . . . .16PHC1116 . . . . . . . . . . . .16PHC1123 . . . . . . . . . . . .16PHC1124 . . . . . . . . . . . .16PHC1154 . . . . . . . . . . . .16

PHC1174 . . . . . . . . . . . .17PHC1204 . . . . . . . . . . . .16PHC1234 . . . . . . . . . . . .17PHC1244 . . . . . . . . . . . .17PHC1254 . . . . . . . . . . . .17PHC1264 . . . . . . . . . . . .17PHC1331 . . . . . . . . . . . .15PHC1333 . . . . . . . . . . . .15PHC1335 . . . . . . . . . . . .15PHC1336 . . . . . . . . . . . .15PHC1341 . . . . . . . . . . . .17PHC1343 . . . . . . . . . . . .17PHC1344 . . . . . . . . . . . .17PHC1346 . . . . . . . . . . . .17PHC1364 . . . . . . . . . . . .17PHC1431 . . . . . . . . . . . .15PHC1433 . . . . . . . . . . . .15PHC1434 . . . . . . . . . . . .15PHC1471 . . . . . . . . . . . .15PHC1473 . . . . . . . . . . . .15PHC1474 . . . . . . . . . . . .15PHC1506 . . . . . . . . . . . .17PHC1514 . . . . . . . . . . . .15PHC1524 . . . . . . . . . . . .15PHC1536 . . . . . . . . . . . .16PHC1544 . . . . . . . . . . . .17PHC1564 . . . . . . . . . . . .15PHC1574 . . . . . . . . . . . .16PHC1585 . . . . . . . . . . . .15PHC1594 . . . . . . . . . . . .15PHC1601 . . . . . . . . . . . .17PHC1603 . . . . . . . . . . . .17PHC1604 . . . . . . . . . . . .17PHC1615 . . . . . . . . . . . .15PHC1624 . . . . . . . . . . . .15PHC1625 . . . . . . . . . . . .17PHC1634 . . . . . . . . . . . .17PHC1674 . . . . . . . . . . . .15PHC1684 . . . . . . . . . . . .17PHC1691 . . . . . . . . . . . .16PHC1694 . . . . . . . . . . . .16PHC1804 . . . . . . . . . . . .17PHC2011 . . . . . . . . . . . .16PHC2013 . . . . . . . . . . . .16PHC2015 . . . . . . . . . . . .16PHC2031 . . . . . . . . . . . .16PHC2033 . . . . . . . . . . . .16PHC2035 . . . . . . . . . . . .16PHC2054 . . . . . . . . . . . .15PHC2111 . . . . . . . . . . . .17PHC2113 . . . . . . . . . . . .17PHC2115 . . . . . . . . . . . .17PHC2116 . . . . . . . . . . . .17PHC3011 . . . . . . . . . . . .17PHC3013 . . . . . . . . . . . .17PHC3015 . . . . . . . . . . . .17PHC3015L . . . . . . . . . . .17PHC3016 . . . . . . . . . . . .17PHC4014 . . . . . . . . . . . .16PHC4031 . . . . . . . . . . . .16PHC4033 . . . . . . . . . . . .16PHC4044 . . . . . . . . . . . .16PHC4054 . . . . . . . . . . . .16PHC4064 . . . . . . . . . . . .16PHC4244 . . . . . . . . . . . .16PHC4814 . . . . . . . . . . . .16PHC5015 . . . . . . . . . . . .17PHC7015 . . . . . . . . . . . .15

PHC7024 . . . . . . . . . . . .17PHC7041 . . . . . . . . . . . .16PHC7043 . . . . . . . . . . . .16PHC7044 . . . . . . . . . . . .16PHC7045 . . . . . . . . . . . .16PHC7056 . . . . . . . . . . . .17PHC7064 . . . . . . . . . . . .17PHC7074 . . . . . . . . . . . .15PHC7075 . . . . . . . . . . . .17PHC7084 . . . . . . . . . . . .16PHC7104 . . . . . . . . . . . .15PHC7141 . . . . . . . . . . . .15PHC7143 . . . . . . . . . . . .15PHC7145 . . . . . . . . . . . .15PHC7146 . . . . . . . . . . . .15PHC7204 . . . . . . . . . . . .15PHC7914 . . . . . . . . . . . .15PHC8011 . . . . . . . . . . . .15PHC8013 . . . . . . . . . . . .15PHC8014 . . . . . . . . . . . .15PHC8015 . . . . . . . . . . . .15PHC8021 . . . . . . . . . . . .17PHC8023 . . . . . . . . . . . .17PHC8024 . . . . . . . . . . . .17PHC9151 . . . . . . . . . . . .16PHC9154 . . . . . . . . . . . .16PHC9171 . . . . . . . . . . . .16PHC9173 . . . . . . . . . . . .16PHC9174 . . . . . . . . . . . .16PHC9211 . . . . . . . . . . . .15PHC9213 . . . . . . . . . . . .15PHC9214 . . . . . . . . . . . .15PHC9215 . . . . . . . . . . . .15PHC9221 . . . . . . . . . . . .16PHC9224 . . . . . . . . . . . .16PHC9241 . . . . . . . . . . . .15PHC9243 . . . . . . . . . . . .15PHC9244 . . . . . . . . . . . .15PHC9251 . . . . . . . . . . . .16PHC9253 . . . . . . . . . . . .16PHC9254 . . . . . . . . . . . .16PHC9255 . . . . . . . . . . . .16PHC9261 . . . . . . . . . . . .16PHC9264 . . . . . . . . . . . .16PHC9271 . . . . . . . . . . . .16PHC9274 . . . . . . . . . . . .16PHC9281 . . . . . . . . . . . .15PHC9283 . . . . . . . . . . . .15PHC9284 . . . . . . . . . . . .15PHC9285 . . . . . . . . . . . .15PHC9291 . . . . . . . . . . . .16PHC9293 . . . . . . . . . . . .16PHC9294 . . . . . . . . . . . .16PHC9295 . . . . . . . . . . . .16PHC9311 . . . . . . . . . . . .16PHC9313 . . . . . . . . . . . .16PHC9314 . . . . . . . . . . . .16PHC9315 . . . . . . . . . . . .16PHC9341 . . . . . . . . . . . .16PHC9343 . . . . . . . . . . . .16PHC9344 . . . . . . . . . . . .16PHC9345 . . . . . . . . . . . .16PHC9381 . . . . . . . . . . . .16PHC9383 . . . . . . . . . . . .16PHC9384 . . . . . . . . . . . .16PHC9385 . . . . . . . . . . . .16PHC9391 . . . . . . . . . . . .17PHC9393 . . . . . . . . . . . .17






PHC9394 . . . . . . . . . . . .17PHC9411 . . . . . . . . . . . .15PHC9413 . . . . . . . . . . . .15PHC9414 . . . . . . . . . . . .15PHC9415 . . . . . . . . . . . .15PHC9431 . . . . . . . . . . . .16PHC9434 . . . . . . . . . . . .16PHC9461 . . . . . . . . . . . .13PHC9461 . . . . . . . . . . . .16PHC9464 . . . . . . . . . . . .13PHC9464 . . . . . . . . . . . .16PHC9471 . . . . . . . . . . . .16PHC9474 . . . . . . . . . . . .16PHC9501 . . . . . . . . . . . .16PHC9504 . . . . . . . . . . . .16PHC9511 . . . . . . . . . . . .14PHC9513 . . . . . . . . . . . .14PHC9514 . . . . . . . . . . . .14PHC9531 . . . . . . . . . . . .14PHC9533 . . . . . . . . . . . .14PHC9534 . . . . . . . . . . . .14PHC9541 . . . . . . . . . . . .14PHC9543 . . . . . . . . . . . .14PHC9544 . . . . . . . . . . . .14PHC9551 . . . . . . . . . . . .15PHC9553 . . . . . . . . . . . .15PHC9554 . . . . . . . . . . . .15PHC9561 . . . . . . . . . . . .14PHC9563 . . . . . . . . . . . .14PHC9564 . . . . . . . . . . . .14PHE0011 . . . . . . . . . . . .17PHE0023 . . . . . . . . . . . .17PHG0014 . . . . . . . . . . . .15PHG0021 . . . . . . . . . . . .12PHG0021 . . . . . . . . . . . .15PHG0021L . . . . . . . . . . .12PHG0021L . . . . . . . . . . .15PHG0023 . . . . . . . . . . . .12PHG0023 . . . . . . . . . . . .15PHG0024 . . . . . . . . . . . .12PHG0024 . . . . . . . . . . . .15PHG0026 . . . . . . . . . . . .12PHG0026 . . . . . . . . . . . .15PHG0035 . . . . . . . . . . . .17PHG0041 . . . . . . . . . . . .17PHG0043 . . . . . . . . . . . .17PHG0044 . . . . . . . . . . . .17PHG0045 . . . . . . . . . . . .17PHG0046 . . . . . . . . . . . .17PHG0051 . . . . . . . . . . . .17PHG0094 . . . . . . . . . . . .15PHG0126 . . . . . . . . . . . .15PHG0134 . . . . . . . . . . . .17PHG0154 . . . . . . . . . . . .15PHG0174 . . . . . . . . . . . .15PHG0184 . . . . . . . . . . . .15PHG0194 . . . . . . . . . . . .15PHG0204 . . . . . . . . . . . .15PHG0214 . . . . . . . . . . . .15PHG0224 . . . . . . . . . . . .15PHG0234 . . . . . . . . . . . .15PHG0244 . . . . . . . . . . . .15PHG0254 . . . . . . . . . . . .16PHG0261 . . . . . . . . . . . 119PHG0261 . . . . . . . . . . . .12PHG0261 . . . . . . . . . . . .15PHG0263 . . . . . . . . . . . .12PHG0263 . . . . . . . . . . . .15

PHG0264 . . . . . . . . . . . .12PHG0264 . . . . . . . . . . . .15PHG0266 . . . . . . . . . . . .12PHG0266 . . . . . . . . . . . .15PHG0271 . . . . . . . . . . . .15PHG0273 . . . . . . . . . . . .15PHG0274 . . . . . . . . . . . .15PHG0276 . . . . . . . . . . . .15PHG0286 . . . . . . . . . . . .15PHG0296 . . . . . . . . . . . .17PHG0311 . . . . . . . . . . . 124PHG0311 . . . . . . . . . . . .15PHG0311L . . . . . . . . . . 124PHG0311L . . . . . . . . . . .15PHG0313 . . . . . . . . . . . .15PHG0314 . . . . . . . . . . . 124PHG0314 . . . . . . . . . . . .15PHG0315 . . . . . . . . . . . 124PHG0315 . . . . . . . . . . . .15PHG9014 . . . . . . . . . . . .15PHG9074 . . . . . . . . . . . 124PHG9074 . . . . . . . . . . . .16PHG9114 . . . . . . . . . . . .17PHG9202 . . . . . . . . . . . .17PHG9203 . . . . . . . . . . . .17PHG9204 . . . . . . . . . . . .17PHG9211 . . . . . . . . . . . .17PHG9214 . . . . . . . . . . . .17PHG9302 . . . . . . . . . . . .17PHG9305 . . . . . . . . . . . .17PHP0024 . . . . . . . . . . . .15PHP0025 . . . . . . . . . . . .15PHP0034 . . . . . . . . . . . .17PHR3015 . . . . . . . . . . . .17PHR3025 . . . . . . . . . . . .17PHR5001 . . . . . . . . . . . .15PHS0044 . . . . . . . . . . . .15PHZ0014 . . . . . . . . . . . .15PMC4051 . . . . . . . . . . . .13PMC4054 . . . . . . . . . . . .13PMG0041 . . . . . . . . . . . 124PMG0043 . . . . . . . . . . . 124PMG0044 . . . . . . . . . . . 124PMG0045 . . . . . . . . . . . 124PRC0011 . . . . . . . . . . . .18PRC0014 . . . . . . . . . . . .18PRC0021 . . . . . . . . . . . .18PRC0024 . . . . . . . . . . . .18PRC0031 . . . . . . . . . . . .18PRC0034 . . . . . . . . . . . .18PRC0051 . . . . . . . . . . . .18PRC0053 . . . . . . . . . . . .18PRC0054 . . . . . . . . . . . .18PRC0061 . . . . . . . . . . . .18PRC0063 . . . . . . . . . . . .18PRC0064 . . . . . . . . . . . .18PRC0104 . . . . . . . . . . . .18PRC0131 . . . . . . . . . . . .18PRC0133 . . . . . . . . . . . .18PRC0134 . . . . . . . . . . . .18PRC0181 . . . . . . . . . . . .18PRC0184 . . . . . . . . . . . .18PRC0811 . . . . . . . . . . . .18PRC0813 . . . . . . . . . . . .18PRC0814 . . . . . . . . . . . .18PRC0815 . . . . . . . . . . . .18PRC1011 . . . . . . . . . . . .18PRC1014 . . . . . . . . . . . .18

PRC1015 . . . . . . . . . . . .18PRC1054 . . . . . . . . . . . .18PRC1066 . . . . . . . . . . . .18PRC1071 . . . . . . . . . . . .18PRC1074 . . . . . . . . . . . .18PRC2011 . . . . . . . . . . . .18PRC2015 . . . . . . . . . . . .18PRC2016 . . . . . . . . . . . .18PRC3013 . . . . . . . . . . . .18PRC3014 . . . . . . . . . . . .18PRC3015 . . . . . . . . . . . .18PRC4014 . . . . . . . . . . . .18PRC4024 . . . . . . . . . . . .18PRC4033 . . . . . . . . . . . .18PRC4034 . . . . . . . . . . . .18PRC4035 . . . . . . . . . . . .18PRC7015 . . . . . . . . . . . .18PRG0111 . . . . . . . . . . . .18PRG0113 . . . . . . . . . . . .18PRG0114 . . . . . . . . . . . .18PRG0115 . . . . . . . . . . . .18PSC0011 . . . . . . . . . . . .19PSC0014 . . . . . . . . . . . .19PSC0021 . . . . . . . . . . . .19PSC0024 . . . . . . . . . . . .19PSC0041 . . . . . . . . . . . .19PSC0044 . . . . . . . . . . . .19PSC0045 . . . . . . . . . . . .19PSC0061 . . . . . . . . . . . .19PSC0064 . . . . . . . . . . . .19PSC0101 . . . . . . . . . . . .19PSC0104 . . . . . . . . . . . .19PSC0151 . . . . . . . . . . . .19PSC0154 . . . . . . . . . . . .19PSC0881 . . . . . . . . . . . .19PSC0884 . . . . . . . . . . . .19PSC2011 . . . . . . . . . . . .19PSC2014 . . . . . . . . . . . .19PSC2015 . . . . . . . . . . . .19PSC3011 . . . . . . . . . . . .19PSC3014 . . . . . . . . . . . .19PSC4031 . . . . . . . . . . . .19PSC4034 . . . . . . . . . . . .19

R–VR210-01 . . . . . . . . . . . 123R210-01 . . . . . . . . . . . 151R250-01 . . . . . . . . . . . 155R250-05 . . . . . . . . . . . 155R705-07 . . . . . . . . . . . .97R7799-105 . . . . . . . . . . 119R790-07 . . . . . . . . . . . .94RBCP10 . . . . . . . . . . . . 183RBCP20 . . . . . . . . . . . . 183RBCS10 . . . . . . . . . . . . 183RBCS20 . . . . . . . . . . . . 183RTCS10 . . . . . . . . . . . . 183SA100-04 . . . . . . . . . . . 108TRCS10 . . . . . . . . . . . . 183TRCS20 . . . . . . . . . . . . 183TRCS30 . . . . . . . . . . . . 183V492-20 . . . . . . . . . . . .97V494-20 . . . . . . . . . . . .97

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14_1 Cultivo Cel Animales Gibco Catalogue Edition 49

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Transcript of 14_1 Cultivo Cel Animales Gibco Catalogue Edition 49