BD Biosciences

January 19, 2011

Advanced Surfaces for Optimizing Cell Growth & DifferentiationPaula Flaherty

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Topics for Discussion

Overview of Cell Culture Surfaces and Representative Applications

• BD Falcon™ Tissue Culture (TC)-Treated

• BD Primaria™

• Extracellular BD Matrigel™ matrix & BD BioCoat™

• BD PureCoat™ Surfaces

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Cell culture systems: 2D 3D

2D2D• Non-treated (hydrophobic)

• BD Falcon™ Tissue Culture (TC) treated [net (-) charge]• BD Primaria™ [mixture of (-) and (+) charge]

• BD BioCoat™ (large variety of ECMs, poly-lysine) • BD PureCoat™ [carboxyl (-), amine (+); animal free]

3D3D• ECM coatings (BD Matrigel™ matrix, BD™ Laminin/Entactin, Collagens)

• Rigid 3D Scaffolds (e.g., PLA, PGA)• Animal-free hydrogels (BD PuraMatrix™ Peptide Hydrogel)

• Cell culture inserts (e.g., co-culture models such as BBB)• Animal models

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Cell Culture Models Support or Promote Cell Behavior

Cell behaviors (differentiation, functionality) influenced by cues in the microenvironment:

• Cell morphology (structure, phenotype)

• Polarity (functional directionality)

• Growth (proliferation)

• Cell motility (migration, invasion)

• Neurite outgrowth

• Signal transduction (surface receptor function)

• Gene and protein expression (different cell types can express different genes/proteins; liver vs. heart vs. brain)

• Biochemical activities (proteins, enzymes)

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Overview of Tissue Culture (TC) Surface Treatment

• Traditional TC-surface chemistry is achieved by placing polystyrene vessels into a vacuum chamber containing a mixture of gases, or by corona discharge

• Under specific conditions, BD creates a vacuum gas plasma that reacts with the polystyrene and alters the surface chemistry

• Used extensively for culturing a large variety of anchorage-dependent cells

hydrophobic hydrophilic, net (-) charge

Vacuum-gas plasma treatment, oxygen Untreated polystyrene

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BD Primaria Cultureware

BD Primaria™ is prepared by treating polystyrene vessels with vacuum-gas plasmamixture containing oxygen and ammonia. The treated surface is comprised of oxygen- and nitrogen-containing functional groups.

Applications:

• Attachment and differentiation of certain fastidious cell types such as primary neurons, hepatocytes, endothelial cells, & cardiomyocytes

• Low to moderate scale for cell expansion (up to 75 cm2 flask)

• Low to moderate throughput for cell-based assays (up to 96-well)

hydrophilic, (-) and (+) charge

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NIH-3T3 Cells Exhibit Enhanced Growth on BD Primaria

BD Falcon™

Non-treated

BD Falcon TC-treated

BD Primaria

NIH-3T3 24 hr post-seeding(in serum-free media)

0

0.2

0.4

0.6

0.8

1

1.2

1.4

1.6

Non-treated TC-treated BD Primaria™

MTS

Sig

nal (

OD

@ 4

90nm

)

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BD Primaria Key References

Perez, RG, et al. (2002) Journal of Neuroscience, Vol. 22, pp. 3090-3099.

BD PrimariaNeuronal cells

Kilic, A, et al. (2010) Cardiovascular Research, doi: 10.1093/cvr/cvq254, published online August 2, 2010.

BD PrimariaCardiomyocytes

Ward, RJ, et al. (2009) Cancer Research, Vol. 69, pp. 4682-4690.

BD PrimariaCancer stem cells

Li, L and Porter, TD (2009) Journal of Biochemical & Molecular Toxicology, Vol. 23, pp. 357-363.

BD Primaria™Hepatocytes

ReferenceSurfaceCell Type

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The Extracellular Matrix

• Complex mixture containing glycoproteins, collagens, and proteoglycans

• Forms structural framework that stabilizes tissues and provides mechanical support for cell attachment

• Plays important role in cell proliferation, migration,shape, orientation, and differentiation (e.g., signal transduction, gene expression, enzymatic activities)

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The Basal Lamina: A Thin ‘Mat’ that Underlies Epithelial Cell Sheets and Tubes

basal lamina = basement membrane

BD Matrigel™ matrix = reconstituted basement membrane

Figure: Molecular Biology of the Cell (3rd Edition)

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ECM Provides a Physiological Growth Substrate

• Although tissue culture plastic is used for many cell types, this environment is not physiological

• ECM-based growth substrates provide a physiological environment that supports and promotes key cell functions

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ECM Contributes to Intracellular Signaling Pathways

• ECM molecules interact with cell surface receptors (e.g., regulation of integrin signaling by fibronectin:integrin interactions)

• The ECM appears to function in the storage and presentation of growth factors

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ECM Components

Glycoproteins

Fibronectin – BD BioCoat™ & vialed

Laminin – BD BioCoat & vialed

Vitronectin – vialed only

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Fibronectin

Figure: Molecular Biology of the Cell (4th Edition)

• Large dimeric protein (multiple isoforms)

• Contributes to matrix organization

• Cell receptors (integrins) bind to FN ‘RGD motif’

• Promotes cell differentiation and functionality (e.g., cell migration, integrin signaling, gene expression)

R

RR

R

RGD

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Laminin

cell binding

cell binding

entactin binding col IV binding

col IV binding

heparin binding

Laminin

• Large heterotrimeric proteins (11 isoforms)

• Primarily found in basal lamina

• Major structural component of basal lamina

• Cell receptors (integrins) bind to multiple sites on LM

• Promotes cell differentiation and functionality (e.g., neurite outgrowth, receptor signaling, gene expression)

Figure: Molecular Biology of the Cell (4th Edition)

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Collagens

• Most ubiquitous ECM molecules (at least 16 types)– subunits of collagen ‘trimer’ encoded by multiple genes

• Fibrous proteins that provide structure and resiliency to tissues

• Major component of skin and bone

• Most abundant protein in mammals (~ 25% of total protein mass)

Type Tissue Distribution

Fibrillar I, V bone, skin, tendon, (polymerized fibrils) cornea, internal organs

II cartilage, notochord

III skin, muscle, blood vessels

Network-Forming IV all basal laminaes

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Collagen Fibrils in Connective Tissue of Skin

Figure: Molecular Biology of the Cell (3rd Edition)

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BD Matrigel™ Matrix: Reconstituted Basement Membrane

Composition

Laminin ~ 60%

Collagen IV ~ 30%

Entactin ~ 8%

Heparan sulfate proteoglycan (perlecan)

Growth factors (e.g., PDGF, EGF, TGF-β)

Matrix metalloproteinases

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BD BioCoat Cultureware

BD BioCoat™ surfaces are prepared using optimized manufacturing processes. Treated surfaces consist of a large variety of extracellular matrix (ECM) proteins and attachment factors.

• Applications:

– Attachment and differentiation of primary cells

– Stem cell proliferation & differentiation

– Improved attachment and growth of transfected cell lines (e.g., HEK-293)

– Enhanced growth and functionality of transformed cell lines

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BD BioCoat™ Cultureware

• Gelatin

• Poly-D-Lysine

• Poly-L-Lysine

• Poly-D-Lysine/Laminin

• Poly-L-Ornithine/Laminin

• Custom coatings

• BD Matrigel™ matrix

• Laminin

• Fibronectin

• Laminin/Fibronectin

• Collagen I

• Collagen IV

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Primary Endothelial Cells Exhibit Enhanced Growth on BD BioCoat Collagen I

BD Falcon™

TC-treated

BD BioCoat™Collagen I

HUVEC Fetal Bovine Heart

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Saline Control 100 µM Glutamate

Rat Cortical Neurons Exhibit Differentiated Morphology and Function on BD BioCoat™ Laminin/Fibronectin

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HTS Analysis of PC-12 Cell Neurite Outgrowth Using the BD Pathway™ Bioimager

Control 200 ng/ml NGF

Neurite Total Length

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40

80

120

160

0.01 0.1 1.0

NGF Dose (µM, on log scale)

Res

pons

e Le

vel

β-tubulin staining, 20x objective

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After Wash, Before Wash After Wash Calcein AM Staining

BD Falcon™

TC-treated

BD BioCoat™Poly-D-Lysine

• 384-well b/c plates; serum-free conditions for 24 hours; washed with 384-well plate washer

• EcoPack2-293: transformed cell line derived from HEK-293 (Clontech)

Transfected EcoPack™2-293 cells Exhibit Strong Adherence to BD BioCoat Poly-D-Lysine

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BD Matrigel Matrix for Feeder-Free hES Culture

hES cells cultured on BD Matrigel™ matrix:

• Maintain normal karyotype

• Demonstrate a stable proliferation rate and high telomerase activity

• Express characteristic undifferentiated hES cell markers

• Form embryoid bodies when transferred to low attachment substrate

• Form teratomas in severe combined immunodeficient (SCID) mice and differentiate into cells from all three germ layers

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Undifferentiated hES Cell Colonies

• Compact and dense H9 colonies on MEF feeders• Spread-out and monolayer-like colonies on BD Matrigel™ matrix

MEF-conditioned mediahES mediaMEF feeder layer BD Matrigel hESC-qualified matrix BD Matrigel hESC-qualified matrix

mTeSR™1

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SSEA-4

OCT-4

BD™ Laminin/Entactin complex BD Matrigel™ hESC-qualified Matrix

H9 Cells Cultured in mTeSR™1 Medium Express Markers Specific for Undifferentiated hES Cells

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Epithelial and Endothelial Cell Differentiation on BD Matrigel matrix (3D) and BD PuraMatrix (3D)

MCF-10A cells form acinar-like morphology on BD Matrigel™ matrix (3D only)*

Endothelial ‘tubes’ formed on BD Matrigel matrix (3D only)

*Debnath J, et al. (2003) Methods, Vol. 30, pp. 256-268.+Wang, S, et al. (2008) Tissue Eng Part A, Vol. 14, pp. 227-236.

Primary rat hepatocyte spheriod on BD PuraMatrix™+

Mammary Epithelial Liver Epithelial Endothelial

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Calcein AM staining, 4x confocal

[Suramin]

HTS Analysis of Endothelial Cell Tube Formation using the BD Pathway™ Bioimager

0 160 μM

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BD BioCoat™, 3D Cell CultureKey References

Cote, MC, et al. (2010) Journal of Biological Chemistry, Vol. 285, pp. 8013-8021.

BD Matrigel Matrix (3D)

Endothelial (tube formation)

Debnath, J, et al. (2003) Methods, Vol. 30, pp. 256-268.BD Matrigel™Matrix (3D)

Mammary epithelial cells

Gupta, MK, et al. (2009) Molecular Pharmacology, Vol. 76, pp. 314-326.

PDL-Laminin (BD BioCoat)

Neuronal cells

Wang, S, et al. (2008) Tissue Engineering Part A, Vol. 14, pp. 227-236.

BD™ PuraMatrix™

(3D)Hepatocytes

Liu, X, et al. (2010) American Journal of Pathology, Vol. 176, pp. 504-515.

Fibronectin(BD BioCoat)

Endothelial progenitors

ReferenceSurfaceCell Type

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• Chemically defined, animal-free surfaces

• Manufactured using a proprietary thin-film coating technology

- BD PureCoat™ amine

- BD PureCoat carboxyl

BD PureCoat Surfaces

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Plasma

Radiofrequency Power Applied to System

Monomer Vapour Flows into Chamber

Plasma Chamber (under vacuum)

Substrate(dish, plate, flask)

The Coating is Applied Using Plasma Polymerization

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BD PureCoat™ Surface Properties

• The amine surface is nitrogen rich, and positively charged

• The carboxyl surface is oxygen rich, and negatively charged

• The thin-film coating is applied to TC-treated vessels

• The detailed chemical composition of the monomers used in the manufacturing process is proprietary

• The charged chemical groups are covalently bound to the plastic

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BD PureCoat™ – Representative Applications

• Cell attachment and proliferation in serum-free or serum-reduced conditions

• Attachment & differentiation of primary neurons andastrocytes

• Attachment, growth, and differentiation of mesenchymal stem cells

• Cell-based HTS assays– GPCR (e.g., HEK-293), cAMP, proteasome-inhibition

• Cell transfection

• Recovery of cells from cryopreservation

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Increased BHK-21 Cell Proliferation in Reduced Serum (1% FBS) on BD PureCoat Amine

Tissue Culture

BD PureCoat™ amine

• > 100% increase in proliferation vs. TC and Competitor C

• 3d growth assay (96-well format), n = 10 wells/surface

0

0.5

1

1.5

2

2.5

3

Rel

ativ

e A

bsor

banc

e U

nits

#1 #3#2#1 #3#2#1 #3#2

TCCompetitor CBD PureCoat amine

6400 cells/well 3200 cells/well 1600 cells/well

Initial seeding density

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Tissue Culture Competitor C BD PureCoat™ carboxyl

Images captured 24h post seeding (100x magnification)

~25-45% increase compared to TC and

Competitor C surfaces

Improved recovery from cryopreservation: LnCAPProstate Cancer Cells on BD PureCoat Carboxyl

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Anti-β-tubulin III Anti-PGP 9.5

Differentiation of Primary Rat CerebellarGranule Cells on BD PureCoat™ Amine

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Differentiation of Rat Cerebellar Granule Cells on BD PureCoat Amine

Rat cerebellar granule (RCG) cells cultured on BD PureCoat™ amine for 24 and 48 hours; immunostained with anti-tubulin IIIβ

• RCG cells exhibit extensive neurite outgrowth• Neurite length increases with time

24 hours post-isolation 48 hours post-isolation

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TC-treated BD PureCoat amine

• Astrocytes were cultured for 24 hours, and then stained with antibody directed against Glial Fibrillary Acidic Protein (GFAP)

Differentiation of Rat Primary Astrocyteson BD PureCoat™ amine

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• 384-well b/c plates; serum-free conditions for 24 hours; washed with 384-well plate washer

• EcoPack™2-293: transformed cell line derived from HEK-293 (Clontech)

Before wash

After wash

Tissue Culture Competitor C BD PureCoat™amine

EcoPack™2-293 cells exhibit strong attachment to BD PureCoat Amine

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Analysis of Adult-Derived Stem Cells

• Human bone marrow-derived mesenchymal stem cells (hMSCs, Lonza)

– Cell source: posterior iliac crest of pelvic bone, normal donors

– hMSC maintenance medium: MSCGM™ (Lonza)

– hMSC differentiation:

• Adipogenic induction medium (Lonza)

• Osteogenic induction medium (Lonza)

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Analysis of hMSC Growth

Methodology

• Prepare cell suspension using MSC growth medium (MSCGM, Lonza)

• Seed 6,000 cells/cm2 on TC-treated and BD PureCoat™ amine 6-well plates

• Culture cells for multiple passages

• Sample analysis:– Growth kinetics: Collect samples at multiple time points (day 1 - day 4)

for each passage number and assess cell confluence using the IncuCyte™ Plus (Essen BioScience)

– Cell yield: Samples from day 4 of each passage number were analyzed using the Vi-CELL™ automated cell counter (Beckman Coulter)

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0

20

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80

100

passage 3

0

20

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80

100

passage 4

day 1 day 2 day 3 day 4

0

20

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passage 2

Donor 2

passage 4

passage 2Donor 1

TC-treated

BD PureCoat™ amine

Mea

n ce

ll co

nflu

ence

(%)/

wel

l

0

20

40

60

80

100

day 1 day 2 day 3

0

20

40

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80

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day 1 day 2 day 3 day 4

Donor 1

passage 2

passage 4

Mea

n ce

ll co

nflu

ence

(%)/

wel

lhMSCs Exhibit an Enhanced Growth Rate on BD PureCoat™ Amine

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hMSCs Exhibit Improved Cell Yields on BD PureCoat Amine

0

200000

400000

600000

800000

1000000

1200000

1400000

1600000

Seed P2 P3 P4 P5

tota

l cel

l yie

ld

Donor 1 Donor 2

0

100000

200000

300000

400000

500000

600000

700000

800000

900000

seed P2 P3 P4 P5 P6

tota

l cel

l yie

ldBD PureCoat Amine Tissue Culture

• hMSCs grow more rapidly on BD PureCoat™ amine in MSCGM medium

- 25-140% increase in yield vs. TC surface

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hMSCs Cultured on BD PureCoat™ Amine Express Characteristic Surface Markers

CD29+

CD44+

CD90+

CD34-

CD45-

Cells (P4) were grown on 6-well plates, collected and resuspended in buffer (DPBS/5% serum), stained with antibodies, and then analyzed using a BD FACSCaliber™

flow cytometer.

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Analysis of hMSC Differentiation

Methodology

Osteogenic lineage:

1. Culture hMSCs (P3) on BD PureCoat™ amine (6-well), and then sub-culture (3,000 cells/cm2) to BD PureCoat amine, BD PureCoat carboxyl, and TC-treated (data not shown) surfaces (6-well)

2. On day 2, induce to differentiate using Osteogenic Induction Medium (Lonza) or feed with MSCGM™ (non-induced control)

3. Re-feed every 3-4 days for 2-3 weeks

4. Wash with 1x PBS, fix with 4% paraformaldehyde, and stain with Alizarin Red

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A. BD PureCoat™ Amine to Amine B. BD PureCoat Amine to Carboxyl

C. Alizarin Red staining

• Both BD PureCoat surfaces support osteogenic differentiation of hMSCs

• Calcium deposits in induced cultures were stained with Alizarin Red dye

Induced

Non-Induced

hMSCs Expanded on BD PureCoat Amine Retain Osteogenic Differentiation Potential

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Analysis of hMSC Differentiation

Methodology

Adipogenic lineage:

1. Culture hMSCs (P4 & P%) on BD PureCoat™ amine or TC-treated surfaces (6-well) and then sub-culture (3,750 cells/cm2) to BD PureCoat carboxyl and TC-treated surfaces (6- or 24-well)

2. At 100% confluence, subject cells to 3 cycles of induction usingAdipogenic Induction Medium and associated protocol (Lonza); non-induced controls fed with MSCGM™ (Lonza) throughout culture period (12-15 days)

3. Wash with 1x PBS, fix with 4% paraformaldehyde, and stain with Oil-Red-O

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BD PureCoat™ amine TC-treated BD PureCoat amine carboxyl TC-treated TC-treated

Indu

ced

Non

-Ind

uced

• Intracellular lipid vacuoles present in induced cultures- stained with Oil-Red-O

hMSC Expanded on BD PureCoat Amine Retain Adipogenic Differentiation Potential

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BD PureCoat Application Notes

#470: Advanced Cell Culture Surfaces: BD PureCoat surfaces provide improved cell attachment and growth of many cell types compared to standard TC vessels

Carboxyl (6-, 24-, 96-well plates)

Carcinoma cells: LNCaP, HT-1080, HepG2

#483: Increased cell yields of LNCaP, BHK-21, and MRC-5 cells on BD PureCoat surfaces

Carboxyl (flasks)

LNCaP, MRC-5 (1o

lung), BHK-21

#485: BD PureCoat surfaces support growth, expansion, and differentiation of stem cells

Amine & Carboxyl

Stem cells: MSCsand ASCs

#471: BD PureCoat Amine supports cell attachment and differentiation of neural cells

AminePrimary neuronal cells

#466: BD PureCoat™ surfaces: Development of two novel culture surfaces that enhance cell performance in cell-based assays

AmineTransfected HEK-293

BD Application NotesSurfaceCell Type

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Uncoated Collagen I PDL

Immunofluorescence Analysis of EcoPack™ Cells (derived from HEK-293) Stained with Integrin αv

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Questions?

Contact information:Paula Flahertye-mail: paula_flaherty@bd.com

Technical Support:tel: 877.232.8995e-mail: labware@bd.combdbiosciences.com/webinarsFor research use only. Not intended for use in diagnostic or therapeutic procedures. BD, BD Logo, and all other trademarks are property of Becton, Dickinson and Company. ©2010 BD