XFp Intro Training Manual Cell Characterization & Stress Tests

XFp Intro Training Manual

Cell Characterization & Stress Tests

XFp Analyzer Consumables

XFp Stress Test Kits

Part # Description # Miniplates

103010-100 XFp Cell Mito Stress Test Kit 6

103017-100 XFp Glycolysis Stress Test Kit 6

XFp FluxPak (contains cartridges, miniplates, and calibrant)

Part # Description # Cartridges

103022-100 XFp FluxPak 12 XFp Cell Culture Miniplates

Part # Description # Miniplates

103025-100 XFp Cell Culture Miniplates 12

Reagents, Calibrant, Medium & Carrier Tray

Part # Description Use/Quantity

102504-100 XF Plasma Membrane Permeabilizer 60 assays

103059-000 XF Calibrant 1 x 100mL bottle

103193-100 XF Base Medium 1 x 100mL bottle

103057-100 XFp Carrier Tray Pack of 4

XFp Port Layout

XFp AnalyzerQuick Info

A B

C D

Instrument Serial #

CONTACT SEAHORSE BIOSCIENCE Need to order consumables? Go to: www.seahorsebio.com/order

Need technical support? Go to: www.seahorsebio.com/techsupport

XFp Analyzer

XFp MINIPLATERecommended initial assay volume: 150-250 µL per well.When performing the medium exchange, ensure that the resulting volume of assay medium allows for injection volumes. The final volume in the wells during the assay must not exceed 275 µL.

XFp INJECTION PORTSRecommended loading volume: 20 - 30 µL(Do not exceed 30 µL due to the risk of contaminating the instrument)

Ensure that all injection ports used in the assay protocol are loaded with an equal volume to prevent incomplete injections. (i.e., if injecting compound through ports “A” and “B”, all “A” and “B” ports must be loaded with either compound or medium).

EXAMPLE VOLUMES FOR AN XF ASSAY

Number of compound injections in assay 1 2 3 4

Assay Medium in wells after medium exchange 150-250 µL 150-225 µL 150-200 µL 150-175 µL

Injection Port A 25 µL 25 µL 25 µL 25 µL

Injection Port B 0 25 µL 25 µL 25 µL

Injection Port C 0 0 25 µL 25 µL

Injection Port D 0 0 0 25 µL

Total volume per well 150-275 µL

XFp Recommended Assay Volumes

Corporate HeadquartersSeahorse Bioscience Inc.16 Esquire RoadNorth Billerica, MA 01862 USPhone: +1-978-671-1600 800-671-0633www.seahorsebio.com

European HeadquartersSeahorse Bioscience EuropeSymbion Science Park Fruebjergvej 3 2100 Copenhagen Denmark

Asia-Pacific HeadquartersSeahorse Bioscience Asia199 Guo Shou Jing Road Suite 207Pudong, Shanghai 201203 CN

How does a Seahorse XF Analyzer work? Take 15 minutes to watch these six videos and you’ll have a good foundation for understanding XF Assays.

Introductory videos available at http://www.seahorsebio.com/learning/videos.php

a. Energy Pathways (2:06 minutes) b. Transient Microchamber (2:12 minutes) c. Drug Injection Ports (1:51 minutes) d. Validation 1 (2:23 minutes) e. Validation 2 (1:19 minutes) f. Measure Glycolysis (2:58 minutes)

Download software to your laptop or desktop: http://www.seahorsebio.com/support/software/update.php

Pre-training

Contact Seahorse Technical Support: www.seahorsebio.com/techsupport

Basic Procedure

XF Assay Roadmap

Planning Your Assay Day Prior to Assay

Day of Assay

After Your Assay

After Your Assay

• Obtain materials • Reconstitute kits • Review media

requirements

• Hydrate cartridge

• Seed cells • Prepare assay

medium • Wash cells • Prepare assay

compounds • Load cartridge

with compounds • Design/load

instrument protocol

• Run assay

Data interpretation

Next experiments

Prepare and run

assay

Assay Workflow

Prepare cells in XFp Cell Culture Miniplate

Prepare sensor cartridge

Hydrate sensor cartridge overnight

3. Begin Assay: Calibrate sensors

4. Add cells to Analyzer

(after calibration)

2. Add compounds to reagent ports

Day before Assay

Day of Assay

1. Change medium to low-buffered, bicarbonate-free assay medium

Basic Procedure

The Well-Equipped XF Assay Laboratory

Extracellular Flux assays, like most live-cell based assays, benefit from a well-equipped tissue culture laboratory. In addition there are certain unique aspects of Seahorse XF assays that require some pieces of equipment not routinely found in a tissue culture lab. This document outlines requirements and suggestions for equipment and supplies to have available when setting up and performing XF assays. For Seahorse products designed for your particular XF instrument, please refer to the accompanying “Know Your Seahorse” document.

Required

• Non-CO2 incubator (37°C) • pH meter • Tissue culture microscope • Cell counter/hemocytometer • P20 and P200 and P1000 pipettes and tips

o Recommended Pipettes and Tips: � Biohit Proline Plus (10-100 µL) OR BioPette Plus (20-200 µL) with:

• Biohit Optifit Tip (catalog # 790351) or • VWR Ultrafine Flextop Tips (Catalog # 37001-532 (USA); 7320504 (EU)) or • Rainin 250 µL Tips (Catalog # RT-205)

Recommended

The following materials are recommended to help you achieve optimal results. The use of each item is outlined in the Basic Procedures associated with each instrument.

• Internet access (required for remote support of instrument, file sharing) • Centrifuge with plate adaptors for cell culture plates (required for suspension cells) • Balance suitable for plates (required for centrifuging suspension cells) • Multichannel pipette (200 µL capacity; 1200 µL capacity useful for 24 well plates) • Water bath set to 37°C • Microcentrifuge • Touch vortex • Reagent reservoirs (useful for using multichannel pipettes) • 15 mL conical tubes • 1.5 mL Eppendorf tubes • Sterile filter (0.22 µm) and bottle or tube for sterilizing media

Seahorse Technical Support: www.seahorsebio.com/techsupport

Basic Procedure

Hydrating a Sensor Cartridge for the XFp Analyzer

Introduction

A critical component of the XF assay platform is the sensor cartridge. Each probe tip of the sensor cartridge is spotted with a solid-state sensor material that detects changes in both pH and O2 concentration over time to calculate rates. In order for the sensors to function correctly, they must be thoroughly hydrated. The following procedure is designed to hydrate and prepare the sensor cartridges for the assay.

Materials

XFp FluxPak (Part # 103022-100), contains: 1. Green Box:

a. Sensor Cartridge (12x) b. Utility Plate (12x) c. Cartridge Lid (12x)

2. Blue Box: a. XFp Miniplate with lid (12x)

3. XF Calibrant (100 mL)

Additional requirements: non-CO2 incubator

Procedure

1. Remove a three-pack of cartridges from the green box, fold it back and forth along the perforation a few times then pull the tubs apart. Remove the foil seal from the tub(s) that will be used.

2. Separate the Utility Plate and Sensor Cartridge (Figure 1). Place the Sensor Cartridge upside down on the lab bench (Figure 2).

Figure 1 Sensor Cartridge being lifted from Utility

Plate.

Figure 2 Utility Plate (left) next to upside

down Sensor Cartridge (right).


Basic Procedure

3. Fill each well of the Utility Plate with 200 µL of XF Calibrant. See table below.

4. Fill the moats around the outside of the wells with 400 µL per chamber. See table below.

5. Return the XFp Sensor Cartridge to the Utility plate that now contains calibrant. 6. Place the cartridge assembly in a non-CO2 37°C incubator overnight. To prevent evaporation of the

XF Calibrant, the incubator should be humidified.

XFp Carrier Trays are included with each instrument. These carriers can hold two (2) XFp cartridge/ miniplate assemblies or three (3) miniplates without cartridges. They provide easier handling and incubation of plates and cartridges as well as compatibility with microplate-based equipment.

Plate Compartment single channel pipette setting 8-channel pipette setting

Wells 200 µL, 8 dispenses 200 µL, 1 dispense

Moat chambers 400 µL, 8 dispenses 200 µL, 2 tips per chamber, 2 dispenses

Figure 3. XFp Carrier tray holding 2

cartridges supported by miniplates.


Basic Procedure

Seeding Adherent Cells in XFp Cell Culture Miniplates Introduction

XF assays are performed in a Seahorse XFp miniplate in conjunction with an XFp sensor cartridge. Each miniplate is formatted as a column of a typical 96-well plate. The seeding surface of each well is 0.106 cm2, approx. 40% the surface area of a standard 96-well plate. This procedure describes recommendations for seeding adherent cells for use with the XFp Analyzer.

XFp miniplate carriers are included with each instrument and available separately. These carriers are designed to hold 3 XFp miniplates and provide easier handling and manipulation of the plates while in the tissue culture hood or cell culture incubator. They are also compatible with microplate centrifuge adapters and most plate readers. The procedure given below can be performed with the XFp miniplate inserted either in the carrier or on its own.

Procedure

1. Remove a three-pack of miniplates from the blue box, fold it back and forth along one of the perforations a few times, and then pull the tubs apart. Remove the foil seal from the tub(s) that you will be using.

2. Add sterile water or PBS to the moat around the cell culture wells. Use an 8-channel pipettor set to 200 µL, and fill both sides of the moat (two tips will fit into each chamber). If no multi-channel pipette is available, fill each chamber of the moat with 400 µL of sterile water or PBS (total 3200 µL).

3. Add 80 µL of assay media only (no cells) to wells A and H. These are background correction wells.

4. Determine the desired seeding concentration. Consult the literature1 and/or test different cell seeding densities to ensure good results. Optimal cell seeding numbers can vary widely but are typically between 5 x 103 and 4 x 104 cells per well. Cells are seeded in 80 µL of growth media.

Example: One wants to seed at 2.0 x 104 cells per well. Thus, the target cell seeding concentration is 2.0 x 104 cells / 0.080 mL = 2.5 x 105 cells per mL.

1 Cell Line Reference Database: http://www.seahorsebio.com/learning/cell-line.php

Diagram of XFp Miniplate highlighting moat with 8 chambers (green) and 6 assay wells (pink). The background wells are not colored.


Basic Procedure

5. Harvest the cells using standard procedures. Resuspend the cells in prepared assay medium, count them, and then dilute to the desired seeding concentration. See example below.

Example: Following harvesting and counting, the cell concentration is 1.6 x 106 cells per mL. To achieve the desired seeding concentration, the dilution factor is 1.6 x 106 cells per mL/2.5 x 105 cells per mL = 6.4. Combine 100 µ L of cells with 540 µ L of growth medium (or other proper ratio of volumes).

6. Add 80 µL of cell suspension to wells B-G (as shown in the figure)

7. Allow the cells to grow overnight in a cell culture incubator. For cells being cultured for longer periods, make sure that the moat does not dry out. Replenish fluids whenever you do a medium exchange on the cells.

8. Check the growth and health of cells using a microscope. Do NOT add fluid to the moat prior to running the assay. It is not necessary to remove fluid from the moat chambers prior to the run.


Basic Procedure

Seeding Suspension Cells in XFp Cell Culture Miniplates

Introduction

XF assays are performed in a Seahorse XFp miniplate in conjunction with an XFp sensor cartridge. Each miniplate is formatted to be a column of a typical 96-well plate, as shown. The seeding surface of each well is 0.106 cm2 – approximately 40% of the bottom surface area of a standard 96-well plate. This procedure describes seeding suspension cells for use with the XFp Analyzer.

Because the measurement of oxygen consumption rates (OCR) and extracellular acidification rates (ECAR) takes place in the microchamber formed at the bottom of the specialized XFp miniplate, suspension cells should be adhered to the bottom of the plate. XFp miniplates can be centrifuged in a standard microplate centrifuge using the XFp miniplate carriers (see next page).

Procedure

1. Remove a three-pack of miniplates from the blue box, fold it back and forth along one of the perforations a few times, and then pull the tubs apart. Remove the foil seal from the tub(s) that you will be using.

2. Some cell lines require a plate coating in order to adhere. Plate coating examples include Cell-Tak, poly-D-lysine, and gelatin. The plate coating will depend on the specific cell type being used in the assay.

3. Determine the optimal seeding concentration. Consult the literature1 and/or test different cell seeding densities to ensure good results. For suspension cells, the optimal seeding density is typically between 1x105 and 4x105 cells per well).2

4. Add 150 µL assay medium (no cells) to wells A and H. These are the Background Correction wells.

1 Cell Line Reference Database: http://www.seahorsebio.com/learning/cell-line.php 2 Note that these values are larger than for adherent cells because (1) suspension cells are often much smaller than adherent cells and (2) they usually have significantly lower rates of basal or resting respiration.

Diagram of XFp Miniplate highlighting moat with 8 chambers (green) and 6 assay wells (pink). The background wells are not colored.


Basic Procedure

5. Harvest and dilute the cells in assay medium3 to the desired concentration, where: desired # cells/well x 20 = # cells/mL

6. Add 50 µL of cell suspension to each well B through G; do not add cells to the Background Correction wells (wells A and H).

7. Place the miniplate(s) in a carrier tray and centrifuge at 300 x g for 1 min with no brake. XFp miniplate carriers are included with each instrument when purchased, and also available separately. These carriers are designed to hold up to 3 miniplates, and fit standard microplate centrifuge adapters. Ensure that the centrifuge rotor is balanced appropriately.

8. Use a microscope to visually confirm adherence of the cells to the well bottom. 9. Taking care not to disturb the cells on the bottom, gently add assay medium to each well to the

desired initial assay volume (usually 180 µL). More details on volumes for use with XF Assay Kits can be found in the XF Stress Test User Guides.

10. Add sterile water or PBS to the moat around the cell culture wells, 100 µL per chamber4. Using an 8-channel pipettor (if available) set to 50 µL, fill both sides of the moat using two tips per chamber. If no multi-channel pipette is available, individually fill each chamber of the moat with 100 µL of sterile water or PBS (total 800 µL).

11. Place the XFp miniplate(s) in a carrier tray in a 37°C non-CO2 incubator for 30 minutes to equilibrate the temperature.

3 The choice of assay medium depends on the type of experiment being performed. Please see the basic procedure “Preparation of Assay Media” for further details. 4 For cells that are plated the same day as the assay is run, just a small amount of fluid in the moat is recommended. For cells being incubated overnight prior to assay (such as adherent cells) it is necessary to fill the moat with 400 µL per chamber to avoid assay well evaporation and edge effects.


Basic Procedure

Preparation of Assay Media for Use in XFp Assays

This basic procedure details the preparation of assay media for use with the (1) XFp Cell Mito Stress Test Kit, and the (2) XFp Glycolysis Stress Test Kit

Seahorse recommends the use of non-buffered assay medium for XF assays to ensure accurate, functional measurements of metabolic activity in an ambient environment. The assay medium recipes for the XF Cell Mito Stress Test and the XF Glycolysis Stress Test are different. Both can be prepared starting with the XF Base Medium and adding different substrates as determined by your cell type and/or assay design. Substrate requirements1 are cell-type and assay-design specific and may need to be determined empirically. For the XF Cell Mito Stress Test (see procedure 1 on next page), assay medium supplemented with glucose, sodium pyruvate and glutamine is recommended. For the XF Glycolysis Stress Test (see procedure 2 on page 4), assay medium supplemented with glutamine is recommended.

Equipment Required: • Water bath set to 37°C

• 0.2 m Sterile Filter and 10 mL syringe

• pH meter

• 1 N NaOH

Reagents Required (depending on the assay to be run):

Source Cat #

XF Base Medium, 2 x 500 mL Seahorse Bioscience 102353-100 XF Base Medium, 100 mL Seahorse Bioscience 103193-100 Glucose (2.5 M) Sigma G8769 Sodium Pyruvate (100 mM solution) Sigma S8636 Sodium Pyruvate (powder) Sigma P5280 L-Glutamine (200 mM solution) Life Technologies 25030-081

1 Glucose, glutamine, and pyruvate are the most commonly added substrates in cell culture medium and their consumption is required for both mitochondrial respiration and glycolysis. In the XF Stress Tests, substrates are usually provided in saturating concentrations to ensure that they are not limiting the rates of respiration (OCR) and/or extracellular acidification (ECAR). Seahorse suggests using assay medium containing 10 mM glucose, 1 mM pyruvate and 2 mM glutamine when first performing the Cell Mito Stress Test and 2 mM glutamine only (no glucose or pyruvate) for the Glycolysis Stress Test assay medium.


Basic Procedure

1. XF Cell Mito Stress Test Assay Medium

Cell Mito Stress Test medium generally contains the substrates glucose, glutamine, and pyruvate. For one XFp miniplate, 10 mL of assay medium is sufficient.

Method

1. Warm 10 mL XF Base Medium to 37°C. 2. Add glucose to the desired final concentration using the following table. Seahorse recommends

using the same concentration of glucose as normally used in the growth medium or use 10 mM glucose as a starting point.

Desired glucose concentration Volume of 2.5M glucose solution per 10 mL

2.5 mM 10 µL 5.5 mM 22 µL 10 mM 40 µL 25 mM 100 µL

3. Add the desired amount of sodium pyruvate using liquid or powder using one of the following tables. Seahorse recommends using the concentration of pyruvate normally found in your growth medium or use 1.0 mM as a starting point. Addition of sodium pyruvate from 100 mM solution:

Desired pyruvate concentration Volume of 100 mM pyruvate solution per 10 mL 0.5 mM 50 µL 1.0 mM 100 µL 2.0 mM 200 µL

For desired pyruvate concentrations greater than 2 mM, using sodium pyruvate powder is recommended to prevent excessive dilution of the assay medium:

Desired pyruvate concentration milligrams of sodium pyruvate per 10 mL medium 5.0 mM 5.5 mg

10.0 mM 11.0 mg

4. Add the desired amount of L-glutamine using the following table. Seahorse recommends using the concentration of glutamine normally found in your growth medium or use 2 mM as a starting point.

Addition of L-glutamine from a 200mM solution:


Basic Procedure

Desired glutamine concentration Volume of 200 mM glutamine solution per 10 mL 1 mM 50 µL 2 mM 100 µL 4 mM 200 µL

5. Adjust pH to 7.4 ± 0.1 using 1 N NaOH. Note: the assay medium is non-buffered, so use small volumes and add slowly to adjust the pH.

6. Sterilize the medium by passing through a 0.2 m filter. Keep the XF Cell Mito Stress Test assay medium at 37°C until ready to use. Important: Seahorse recommends incubating assay medium for less than 4 hours at 37oC as substrates such as glutamine can degrade.

2. XF Glycolysis Stress Test Assay Medium

Glycolysis Stress test assay medium generally contains only glutamine as a substrate. For one cell miniplate, 10 mL of assay medium is sufficient.

Method

1. Warm 10 mL of XF Base Medium to 37°C. 2. Add the desired amount of L-glutamine according to table below. Seahorse recommends using the

concentration of glutamine normally in your growth medium, or use 2 mM as a starting point.

Final glutamine concentration Volume of 200 mM L-glutamine solution per 10 mL 1 mM 50 µL 2 mM 100 µL 4 mM 200 µL

3. Adjust the pH to 7.4 ± 0.1 using 1 N NaOH. Note: the assay medium is non-buffered, so use small volumes and add slowly to adjust the pH.

4. Sterilize the medium by passing through a 0.2 µm filter. 5. Keep the XF Glycolysis Stress Test assay medium at 37°C until ready to use. Important: Seahorse

recommends incubating assay medium for less than 4 hours at 37oC as substrates such as glutamine can degrade.


Basic Procedure

Washing Cells in XFp Cell Culture Miniplates

Before performing an XF assay, growth medium must be replaced with a suitable assay medium (generally this means medium without bicarbonate buffer or serum and with low phenol red content). This procedure describes replacing the growth medium with assay medium for adherent cells grown in XFp cell culture miniplates prior to being assayed using an XFp Analyzer.

Materials Required:

• Prepared assay medium. See the Basic Procedure for Assay Media Preparation for details on choosing and preparing the assay medium.

• Multi-channel pipette, 200 µL capacity, with matching tips

• Tissue culture microscope

• Non-CO2 incubator

Procedure:

1. Warm the assay medium to 37oC. 2. Retrieve the XFp cell miniplate(s) from the tissue culture incubator. You may wish to keep the

miniplate(s) in the Carrier Tray for ease of handling. 3. Look at the cells under the microscope to:

a. Confirm cell health, morphology, and purity (no contamination). b. Ensure that the cells are adhered, and appear as a consistent monolayer. c. Make sure that the background wells (A and H) contain no cells.

4. Wash the cells with assay medium: a. Remove all but 20 μL of the culture medium from each well. The small

amount of medium is left to keep the cells from drying out. b. Gently add approximately 200 µL of assay medium, then remove the

same amount. c. Repeat step 4b, removing all but 20 μL (as in step 4a). d. Add assay medium to a total volume of 180 µL – or the volume

recommended by the particular assay protocol you are using. 5. Observe the assay wells under the microscope to ensure that cells were not

washed away. 6. Place the plate in a 37°C incubator without CO2 for one hour prior to the assay.

Incubating the cell plates without CO2 allows outgassing from the plate and is required for accurate ECAR measurements.


Basic Procedure

Loading the XFp Sensor Cartridge with Compounds Introduction

A key feature of the XFp Analyzer is its ability to inject compounds during the assay and see results in real time. This is accomplished by dispensing compounds that have been loaded into injector ports within the cartride prior to placement in the instrument. This procedure describes the loading processand is intended for use following overnight cartridge hydration. XFp Carrier Trays are included with each instrument. These carriers can hold two (2) XFp cartridge/ miniplate assemblies or three (3) miniplates without cartridges. They provide easier handling and incubation of the cartridge while in a non-CO2 incubator or while loading the cartridge on a lab bench. The procedure given below can be performed with or without the XFp sensor cartridge inserted in the carrier.

Best Practices for Successful Compound Loading

1. Each series of ports must contain the same volume (For example, all A ports must be filled with the same volume; all B ports must be filled with the same volume, etc.).

2. All ports of a given letter designation, including ports belonging to Background Correction or blank wells, need to have liquid loaded into them used to ensure proper injection in all wells.

3. Compounds should be diluted in prepared and pH-adjusted assay medium before being loaded into the sensor cartridge. Refer to the document “Media Preparation” for details. Remember: No BSA or serum in the ports!

4. The hydrated XFp sensor cartridge must remain in the utility plate, and be placed flat on the work surface throughout the loading procedure. Do not lift or angle the plate/cartridge away from the work surface while loading.

5. Handle the XFp cartridge very carefully. Use the Carrier Tray for handling and incubating the sensor cartridge prior to placement in the instrument. Avoid carrying the cartridge long distances. To mitigate the accidental discharge of compounds prior to starting the assay, it is best to load the injection ports while in close proximity to the the XFp Analyzer.

Figure 1. XFp Carrier tray holding 2

cartridges supported by miniplates.


Basic Procedure

Port Loading Procedure

Before starting the compound loading process, have the compounds prepared and a plan for which compounds go into which ports. The recommended injection volume range for the XFp cartridge is 20-30 µL.

Note: The hydrated XFp assay cartridge must remain in the utility plate and be placed flat on the work surface throughout the loading procedure. Do not lift or angle the plate away from the bench while loading. Hold the base of the utility plate whenever handling the cartridge to avoid triggering discharge from the injection ports.

STEP 1 After preparation of compounds in the assay medium of choice, warm them to to 37oC. NOTE: It is strongly recommended that injected compounds be at pH 7.35 - 7.4 at 37°C prior to loading into the injection ports.

STEP 2

Orient the XFp Assay Cartridge: Place well labels (letters A-H) to the left. The triangular notch will be in the bottom left-hand corner. See Figure 2.

STEP 3

Using a p100 or p200 µL pipette1, make sure the tip is securely fitted onto the pipette and aspirate the desired volume of compound to be injected. Note: See recommendations for pipettes and tips on the next page.

STEP 4

Position the pipette tip (filled with your compounds for injection) into the desired port- see diagram in Figure 3.

1 Note: Automated pipettes are generally not recommended for cartridge loading, as they may lead to compound leakage through the bottom of the ports.

Figure 2. Diagram of the

XFp sensor cartridge.


Basic Procedure

Orient the tip at a very slight angle (<5o) and lower the tip as far as it will go without resistance into the port. Do not force the tip into the port.

STEP 5

Dispense the compound solution into the port gently. Withdraw the tip from the port carefully. Avoid creating air bubbles. Important! Do NOT tap any portion of the cartridge in an attempt to alleviate air bubbles. This may cause compound leakage from the injection port. STEP 6 Position yourself at eye level with the cartridge and visually inspect the injection ports for even loading. Make sure there are no residual drops on top of the cartridge. Record the position of any ports which appear uneven for later data analysis. Once all compounds have been loaded according to your experimental design, carefully transfer the cartridge/utility plate assembly to the XFp Analyzer to start calibration..

IMPORTANT: Remove the plate lid before inserting the cartridge into the XFp Analyzer.



Basic Procedure

New Cell Line Characterization To effectively examine bioenergetic function using the Seahorse XFp Extracellular Flux Analyzer, it is essential to first characterize a specific cell type with respect to its metabolic activity under basal and test conditions. The XFp Cell Mito Stress Test and XFp Glycolysis Stress Test Kits can be used in tandem to characterize and phenotype the cell line/type of interest. This method is designed to allow phenotyping of a single sample type in just 2 XF experiments. The first is used to identify the FCCP concentration that provides maximal mitochondrial respiration, and the second uses the information from the first experiment to perform the XFp Cell Mito Stress Test and the XFp Glycolysis Stress Test. In addition, these experiments indicate what adjustments, if any, should be made to the assay conditions to produce the most robust assay data. There are two workflow options: (1) Cells that are NOT limited in number, multiple miniplates can be seeded at different densities to reduce the time between experiments and complete the characterization workflow in a single day (Accelerated Workflow). (2) Cells limited in number, an approach where additional cells are prepared after the results of the first experiment are determined is more appropriate (Standard Workflow). Overview Experiment

Step Rationale Accelerated Workflow

Standard Workflow

1

Seed cells at different densities and visually assess confluency; choose a miniplate for the next step.

To generate metabolic rates within the dynamic range of the instrument, cells should be at 50-90% confluency. Visual assessment is a good first approximation of optimal cell density and will be verified by the experiment in Step 2.

Seed 6 miniplates, 2 at each density; hydrate 2 cartridges

Seed 1-3 miniplates with 2-3 different cell densities; hydrate 1 cartridge

2

Perform a titration of FCCP in the presence of 1µM oligomycin.

Cell types vary in their response to the mitochondrial uncoupler FCCP, and at higher doses a reduced response is observed. Therefore, the FCCP dose that generates the maximal OCR must be determined empirically.

Choose 1 plate from above and perform a 5-point titration to generate dose-response data

See Accelerated Workflow

3

Verify chosen cell density and determine optimal FCCP concentration.

Examine the basal and maximal OCR rates; basal rate for cell density and max rate for FCCP concentration

See Accelerated workflow


Basic Procedure

4

Perform the XFp Cell Mito and XFp Glycolysis Stress Tests

Using 3 wells for each assay, it is possible to characterize the cells’ response to a wide variety of conditions and generate the standard parameters of mitochondrial and glycolytic function.

Choose second plate from original 6and proceed immediately to the second assay.

Seed a second plate and hydrate another cartridge; perform the second assay the next day (or when cells are ready).

There are two parameters that which must be empirically determined to properly characterize cellular metabolic function: the cell seeding density and the concentration of FCCP (Carbonyl cyanide 4-(trifluoromethoxy) phenylhydrazone), which is required to stimulate maximal oxygen consumption. Completion of these experiments provides an initial assessment of both the basal and maximal respiration rates of the cells, and verifies whether the chosen conditions provide rates within the dynamic range of the instrument. Step 1 (Experiment 1): Determining the optimal cell seeding density

Optimal cell seeding number varies by cell type, but is typically between 5x103 and 4x104 cells per well. Generally, densities resulting in 50-90% confluency generate metabolic rates in the desirable/dynamic range of the instrument. Use your own experience with this cell type AND consult the literature to determine the seeding density range to be tested.

Figure 1. Schematic of cell seeding strategy prior to the first XF assay. Three miniplates at relative cell densities of 0.5x, 1x, and 2x are prepared and assessed visually. One plate is chosen to proceed to the first XF assay. If performing the Accelerated Workflow, plate 2 miniplates of each cell density.


Basic Procedure

Use the Cell Line Reference Database on the Seahorse Bioscience website to find published papers utilizing your cell type in XF assays (http://www.seahorsebio.com/learning/cell-line.php). 1) Estimate a number of cells based on other experiments performed on this cell type in your own lab.

XFp Miniplates have a well surface area of 40% of a standard 96-well plate, so scale accordingly. Be aware of time between cell seeding and performance of the assay.

2) Choose 3 cell densities to test1. Either cover the range found in the literature or seed the recommended cells/well plus one at 0.5x cells/well and another at 2x cells/well.

3) For each cell density to be tested, seed 1 or 2 miniplate(s) as directed in the appropriate Basic Procedure for either Adherent or Suspension cells.

4) After the cells have been cultured for the appropriate time2, examine the wells of the miniplate under a microscope. Choose the plate that appears to have cells that are adhered, evenly distributed, and at ~60-90% confluency. Note the seeding density of this plate. DON’T WORRY about choosing between 2 miniplates that appear to have confluency in this range- the dynamic range of the instrument can handle a variety of densities as long as they are not over confluent or very sparse.

Step 2 (Experiment 2): FCCP titration Seahorse Bioscience recommends performing a 5-point titration curve to identify the FCCP concentration that yields maximal oxygen consumption rate (OCR). To do this, serial additions of FCCP are made, using the injection ports in the XFp sensor cartridge. The benefit of this approach is that multiple data points for each dose can be generated, thus improving the robustness of the assay compared to a single-dose-per-well scheme. For this strategy, the plate is divided into 2 groups: a low concentration range (0.125, 0.25, 0.50 µM) and a high concentration range (0.50, 1.0, 2.0 µM). Each group is treated first with 1 µM oligomycin3, then three serial injections of FCCP at different concentrations are made. The resulting data set characterizes the cells’ response to five different doses of FCCP.

1 If you do not have enough cells for all the plates, choose fewer densities and/or seed fewer miniplates. For best results with

this cell characterization method, do not seed different densities within the same miniplate. 2 Culture time depends on the cell type and your biological model: adherent vs. suspension, primary vs. transformed, degree

of differentiation required. Consult the literature for details about cell models of interest. 3 Based on extensive research, Seahorse Bioscience has found that 1 µM oligomycin works well for most cell types. Refer to

the Cell Reference Database http://www.seahorsebio.com/learning/cell-line.php) to search for cell types that have been published


Basic Procedure

1. Prepare XF Cell Mito Stress Test Assay Medium and

warm to 37oC. Adjust pH to 7.4 ± 0.1 at 37oC. 2. Retrieve the chosen miniplate from the CO2

incubator. Note the time. 3. View the cells under the microscope to:

a. Confirm cell health, morphology, seeding uniformity and purity (no contamination).

b. Ensure cells are adhered, with a consistent monolayer.

c. Make sure there are no cells in the background correction wells.

4. Wash cells with XF Cell Mito Stress Test Assay Medium. 5. View the cells under the microscope to ensure that

cells were not washed away. 6. Place the plate in a 37°C incubator without CO2 for

one hour prior to the assay. 7. Prepare Stock Compounds:

Important: Use compounds the same day they are reconstituted. Do not refreeze. Discard any remaining compound. a. Remove one foil pouch from the XFp Cell Mito Stress Test Kit box. Keeping the pouch sealed,

allow the compounds to warm to room temperature for approximately 15 minutes. b. Open the pouch and remove the tubes containing oligomycin (blue cap) and FCCP (yellow

cap). The rotenone/antimycin A (red cap) is not used in this experiment. c. Dissolve the contents of each tube with the volume of prepared assay medium specified in

the table below. Narrow P1000 tips are recommended for this4. Gently pipette up and down ~10 times.

Cell Mito Stress Test Kit Component

Volume of Assay Medium

Resulting Stock Concentration

Working (Port) Concentration

Final (Well) Concentration

Oligomycin 252 µL 50 µM 10 1

FCCP 288 µL 50 µM 1.25-10 uM 0.125-2.0 uM

Rotenone/ Antimycin A Mix Not used

8. From the above stock, prepare the working concentration of oligomycin by pipetting 60 µL of

oligomycin stock into 240 µL of assay medium.

4 Narrow p1000 pipette tips are recommended for reconstituting compounds within the tubes provided (e.g. Fisherbrand™

SureOne™ Micropoint Pipet Tips, catalog #: 02-707-402)

Figure 2. Prototypical dose-response curve for FCCP on mammalian cells, generated by the titration method described below.


Basic Procedure

9. Prepare a serial dilution of FCCP in assay medium, as detailed below. a. Set up 4 Eppendorf tubes; add 800

µL assay medium to the first one, and 500 µL assay medium to the other three.

b. Pipette 200 µL of FCCP stock into the first tube (containing 800 µL assay medium). Mix well, then serially dilute 500 µL three times across the remaining tubes. This will yield FCCP ‘working’ concentrations of 10 µM, 5 µM, 2.5 µM, and 1.25 µM.

10. Remove a hydrated cartridge from the non-CO2 incubator. Load each port of the cartridge as outlined below. Following this scheme will ensure final (well) concentrations of 0.125, 0.25, 0.5

µM in the ‘Low Range’ FCCP group and 0.5, 1.0, and 2.0 µM FCCP in the ‘High Range’ FCCP groups. NOTE: Fill the ports of all wells, including those corresponding the background wells, to ensure successful injections.

Figure 4. Diagram of cartridge and compound ports, showing the concentration of FCCP to be loaded in each port. See the table below for port volumes.

Figure 3. Dilution schematic.


Basic Procedure

11. Create or load your assay template on the XFp. 12. On the Run Screen, press Start and follow the prompts to load the cartridge with its utility plate. 13. When prompted by the software, replace the Utility Plate with the XFp Miniplate. Press Continue. 14. When the assay is complete (approximately 1h 45 min), transfer the data to Wave for analysis.

Step 3. Verify Cell Density and Determine Optimal dose of FCCP

Congratulations! You’ve completed your first XF assay. The next step is to examine the data to make some conclusions and plans for the second experiment. Open the results file in Wave and examine the OCR data, which should look similar to the figure below.

This data will allow you to make two assessments: (1) whether the cell density within the desirable range and (Suitable basal rates are often between 20-150 pmol/min.) and (2) what dose of FCCP should be used to provoke maximal respiration in these cells (the lowest FCCP concentration which yields the highest OCR). The chart above shows an example of the effect of the serial injections of FCCP on the oxygen consumption rate (OCR). For the low range FCCP group (blue trace), the OCR increases with increasing concentrations of FCCP. However at higher doses (green trace) the response flattens out. Below are three more examples plotted to show OCR at each FCCP concentration. The graph to the left shows that the OCR response has not reached an apparent maximum and higher concentrations of FCCP should be tested. The middle graph shows a clear example of a single OCR max at 1 µM FCCP. The graph to the right shows a maximum OCR at 1 µM, but the OCR at 0.5 µM is not significantly different because of overlapping error bars. Either 0.5 or 1.00 µM might reasonably be chosen.

Port Designation

Volume per port

Port Concentration in Wells A-D (Low Range)

Port Concentration in Wells E-H (High Range)

Final Well Concentration A-D (Low Range)

Final Well Concentration E-H (High Range)

A (oligomycin) 20 µL 10 µM 10 µM (same) 1 µM 1 µM (same) B (FCCP) 22 µL 1.25 µM 5 µM 0.125 µM 0.5 µM C (FCCP) 28 µL 1.25 µM 5 µM 0.25 µM 1.0 µM D (FCCP) 30 µL 2.5 µM 10 µM 0.5 µM 2.0 µM

Figure 5. Sample data from an FCCP titration performed as described above in an XFp Analyzer. The blue line represents wells in the Low Range group and the green line shows the results for the High Range well group.


Basic Procedure

Step 4 (Experiment 3): Performing the XFp Cell Mito and Glycolysis Stress Tests simultaneously The next experiment that Seahorse recommends is completing a full XFp Mito Stress Test and XFp Glycolysis Stress Test simultaneously on one XFp Miniplate. This allows for full characterization of the mitochondrial and glycolytic parameters of the cell line of interest.

1) Prepare or choose the cell density according to Step 1 and the results of Experiment 1.

Cell type __________ Density ___________

2) Prepare 10 mL each of Glycolysis Stress Test assay medium and Mito Stress Test assay medium according to the kit User Guides and as detailed in the Media Prep Basic Procedure.

3) Prepare Mito Stress Test and Glycolysis Stress Test Compounds according to the corresponding User Guides.

4) Prepare the optimal FCCP concentration as determined in Experiment 1.

Cell type __________ [FCCP] ___________

5) Load the compounds as indicated below. Be certain to load the appropriate ports in all wells, including background wells A and H.

Figure 6. Rate vs. dose plots for FCCP titrations in different cell types. The highest of the 3 rates measured for each dose was plotted against the final concentration of FCCP in each well.


Basic Procedure

6) Initiate the assay via the instrument interface. 7) When the assay is complete, transfer the results to Wave for examination of the relevant

parameters.

XF Glycolysis Stress Test Parameters XF Cell Mito Stress Test Parameters

OCR

(pm

ol/m

in)

TIME (minutes)

Glyco Stress Test

Mito Stress Test

XFp Intro Training Manual Cell Characterization & Stress Tests

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