Seahorse protocol

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Procedures, techniques & resources for a successful Assay

This learning center is designed to introduce you to the Seahorse XF assay workflow, with a focus on procedures and techniques to ensure optimal XF assay performance and results. As you read through each section, the procedures refer to using the Agilent Seahorse XF Real Time ATP Rate AssayCell Energy Phenotype Test to perform initial cell characterization. The techniques described apply to all Seahorse XF Assays, such as seeding adherent cells, loading injection ports, etc. Only the required consumables will vary according to your XF Analyzer model and XF Assay Kit. Select your XF Analyzer using the drop-down menu, then click a section below to display the relevant content for that step of the XF assay workflow.

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1. Gather XF assays materials

2. Prepare for your XF assay

3. Set up your XF assay

4. Run your XF assay

5. Analyze XF assay results

6. Beyond the basics

1. Gather XF assay materials

This section lists materials required to set up your XF assay.

1.1 Required Agilent materials

1.2 Other required materials

1.1 Required Agilent materials

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Seahorse kit

1.2 Other required materials

Other Required Materials

  • 37°C non-CO2 incubator
  • Cell counter/Hemacytometer
  • 37°C water bath
  • Distilled or Sterile H2O
  • Inverted Brightfield Microscope
  • Touch vortex
  • 15 and 50 mL conical tubes
  • P200, P1000, 8 and/or 12 channel P200 pipettes
  • P200, P1000, 8 channel P200 pipettes
  • Reagent Reservoirs

Recommended Materials

  • Centrifuge with adaptors for cell culture plates (required if using suspension cell types)
  • Microcentrifuge
  • Calibrated pH meter*
  • Stir plate*
  • Sterile filter bottles (0.22 μm filter) and cap*
  • 1.0 N NaOH solution*

(* these items are required if using assay media other than Seahorse XF DMEM pH 7.4 or Seahorse XF RPMI pH 7.4)

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2. Prepare for your XF assay

This section focuses on preparation techniques the day before an XF assay, including guidance for choosing cell seeding densities, techniques for seeding adherent cells on XF tissue culture plates and hydrating XF cartridges.

This section focuses on preparation techniques the day before an assay, including guidance for choosing cell seeding densities, techniques for seeding adherent cells on XFp tissue culture plates and hydrating XFp cartridges.

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2.1 Choose Seeding Densitiy

2.2 Seeding Cells

2.3 Hydrate Cartridge

2.3 Hydrate Cartridge

2.4 Design Experiment

2.1 Choose Seeding Densitiy

Basic procedures for choosing cell seeding densities

To effectively examine metabolic and bioenergetic function using your Agilent Seahorse Extracellular Flux Analyzer, Analyzer, it is essential to first characterize a specific cell type with respect to its metabolic activity under basal and maximal respiration (OCR) and extracellular acidification (ECAR). The Seahorse XF Real-Time ATP rate assay Test Kit The Seahorse XFp Real-Time ATP rate assay Test Kit can be used to characterize the cell line/type of interest in a single assay.two short assays.

Optimal cell seeding number varies by cell type, but is typically between 5 x 103 and 4 x 104 cells per well for adherent cells. Suspension cells require higher seeding density from 5 x 104 to 2 x 105 cells per well depending on cell type.1 x 104 and 8 x 104 cells per well. Generally, densities resulting in 50-90% confluency generate metabolic rates in the desirable/dynamic range of the instrument.

Please consult the Agilent Cell Analysis Publication Database and/or the XF Publication database to provide an initial starting point for cell density values specific to your needs.

Seahorse kit

Basic procedures for choosing cell seeding densities

To effectively examine metabolic and bioenergetic function using your Agilent Seahorse Extracellular Flux Analyzer, it is essential to first characterize a specific cell type with respect to its metabolic activity under basal and maximal respiration (OCR) and extracellular acidification (ECAR). The Seahorse Cell Energy Phenotype Test Kit can be used to characterize the cell line/type of interest in two short assays

Optimal cell seeding number varies by cell type, but is typically between 1 x 104 and 8 x 104 cells per well. Generally, densities resulting in 50-90% confluency generate metabolic rates in the desirable/dynamic range of the instrument.

Please consult the Seahorse Cell Reference Database and/or the XF Publication database to provide an initial starting point for cell density values specific to your needs.

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2.2 Seeding Cells

Basic procedures for seeding adherent cells (typically performed the day before the XFp HS Mini assay)

For each density to be tested, seed as directed for adherent cells. View instructions for seeding suspension cells.

Agilent Seahorse XFp HS Mini Assays are performed in an Agilent Seahorse 96-well 24-well 8-well XFp HS Mini Cell Culture MicroplateMiniplate in conjunction with an XFe96 Sensor Cartridge. This procedure describes recommendations for seeding adherent cell types for use with the Agilent Seahorse Analyzer. View instructions for seeding suspension cells.

A method for four different cell densities using an XF96 cell culture plate, XFe96 cartridge and the Seahorse XF Real-Time ATP rate assay kit with an instrument is recommended for an initial assay.

A method for testing four different cell densities and four different FCCP concentrations using two cell culture plates, two cartridges and the XF Cell Energy Phenotype Test Kit with an instrument is recommended for initial assays.

A method for testing four different cell densities using an XF24 Cell Culture Microplate, XFe24 sensor cartridge and the Seahorse XF Real-Time ATP rate assay kit with an instrument is recommended for initial assays.

A method for testing 2-4 different cell densities using an XFp Cell Culture Miniplate, XFp cartridge and the Seahorse XF Real-Time ATP rate assay kit with an XFp instrumentXF HS Mini Analyzer are recommended for initial assays.

This is a suggested XF96 assay plate map for seeding four cell densities:

Assay plate map xf96

Seeding for Cell Density Titration Assay

This is a suggested assay plate map for seeding four cell densities:

Plate map XF24
Plate map design XFe24

Seeding for FCCP Concentration Titration Assay

If you have already performed the cell seeding density assay and/or know the optimal number of cells per well, the FCCP titration assay may be performed using the optimal cell number (1.0 X cells/well) seeded in all wells except Background Correction wells. Otherwise, follow the same instructions for cell seeding and cartridge hydration/preparation as shown below, and use the following suggested plate layout for testing four concentrations of FCCP:

Plate map design XF24

There are two workflow options: (1) For cells that are NOT limited in number, multiple XFp cell culture miniplates can be seeded at different densities to reduce the time between experiments and complete the characterization workflow more quickly (Accelerated Workflow). (2) For cells limited in number, additional cells are prepared after the results of the first experiment are determined (Standard Workflow).

Experiment Rationale Accelerated Workflow Standard Workflow
Seed cells at single or different densities and visually assess degree of cell confluence; choose a miniplate for the next step. To generate metabolic rates within the dynamic range of the instrument, cells should be 50-90% confluent. Visual assessment is a good first approximation of optimal cell density and will be verified in each assay. Seed 1-2 miniplates at 2-4 different densities according to the diagram below. Seed 1 miniplate at a single cell density; hydrate 1 XFp cartridge.
Plate Map XFp
  • Choose 2-4 cell densities to test, based on standard or accelerated workflow described above. Either cover the range found in the references above, or seed the recommended cells/well value (1X) plus 0.5X, 2X and 4X cells per well.
  • Remove a three-pack of miniplates from the blue box.
  • Remove the foil seal from the tub(s) that will be used.
  • 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).
  • Add 80 μL of growth medium only (no cells) to wells A and H. These are background correction wells.
  • Harvest and re-suspend the cells to desired final concentration to seed in 80 μL of growth medium. Optimal cell seeding numbers vary widely, though are typically between 5×103 – 4×104 cells per well and must be determined empirically. (For example, for 1 x 104 cells per well, resuspend cells 1 x 104 per 80 μL = 1.25 x 105 cells per mL)
Seahorse kit

Basic procedures for seeding adherent cells (typically performed the day before the XF assay)

  • A two-step seeding process is recommended when seeding Agilent Seahorse XF24 Cell Culture Microplates. The two-step process produces a consistent and even monolayer of cells.
  • Harvest and re-suspend the cells to desired final concentration to seed in 80 μL of growth medium. Optimal cell seeding numbers vary widely, though are typically between 5 × 103 – 4 × 104 cells per well and must be determined empirically. (For example, for 1 x 104 cells per well, resuspend cells 1 x 104 per 80 μL = 1.25 x 105 cells per mL)
  • Harvest and re-suspend the cells to desired final concentration to seed in 100 μL of growth medium. Optimal cell seeding numbers vary widely, though are typically between 1×104 – 8×104 cells per well and must be determined empirically. (For example, for 2 x 104 cells per well, resuspend cells 2 x 104 per 100 μL = 2.0 x 105 cells per mL)
  • Seed 80 μL of cell suspension per well; do not seed cells in background correction wells (A1, A12, H1, H12). Be sure to put medium only (no cells) in the background correction wells.
  • Seed 80 μL of cell suspension per well in wells B - G, do not seed cells in background correction wells (A and H). Be sure to put medium only (no cells) in the background correction wells.
  • Seed 100 μL of cell suspension per well; do not seed cells in background correction wells (A1, B4, C3, D6). Be sure to put medium only (no cells) in the background correction wells.
  • IMPORTANT: Allow plate to rest at room temperature in the tissue culture hood for one hour.1 This can promote even cell distribution and reduce edge effects for some cell types. Monitor adherence using a microscope.
  • Place the plate in a standard cell culture incubator to allow cells to adhere. This generally takes approximately 1 hour for strongly adherent cells, but may take 5-6 hours for less adherent cell types. Monitor adherence using a microscope.
  • After the one hour rest step, check cells for adherence.
  • If cells are well-adhered, dispense an additional 150 μL of cell growth media to each well (250 µL total), then transfer plate to a standard cell culture incubator.
  • If cells are not well-adhered to the plate, allow an additional 1-5 hours for the cells to firmly attach (in the biosafety cabinet), then add an additional 150 µL of growth media to each well (250 µL total) and transfer plate to a standard cell culture incubator.
  • After cells have adhered, add 150 μl of growth medium to each well, bringing the total volume of medium in the well to 250 μl. When adding medium to the wells, add it slowly to the sides as not to disturb the newly attached cells.
  • Allow the cells to grow overnight in a cell culture incubator. Monitor growth and health of cells using a microscope.

1 Lundholt BK, Scudder KM, Pagliaro L. A simple technique for reducing edge effect in cell-based assays. J Biomol Screen. 2003 Oct;8(5):566-7

Basic procedure for seeding suspension cells (typically performed the day of the XFp HX Mini assay)

Analyzing non-adherent cells (e.g. T cells, leukemia cell line, etc.) using XF technology requires immobilizing cells to the bottom of the wells. This is enabled by a coating with poly D-lysine (PDL) or Cell-Tak to the bottom of each well. Agilent provides ready-to-use PDL-coated XFe96/XF96XFp Cell Culture Microplates. They are validated and recommended for use with T cells in XF T cell activation assay. Prewarm the ready-to-use PDL plates in a 37°C non-CO2 incubator overnight prior to use for seeding cells (minimally 6 hours).

Seeding suspension cells is typically performed on the day of your XF assay, click to view instructions for seeding suspension cells.

If Cell-Tak coating is desired, prepare Cell-Tak coated XFe96/XF96XFp Cell Culture plate(s) as described below:

  • The optimal Cell-Tak solution concentration for Agilent Seahorse Cell Culture MicroplateMiniplate is 22.4 μg/mL.
  • Prepare 2.5 mL1.5 mL0.25 mL of this solution per plate for each assay. Refer to the Manufacturer's protocol to prepare this solution.
  • Apply 2550 μL of the solution to each well for 20 minutes at room temperature.
  • Wash each well twice using 200 μL of sterile water.
  • Cell-Tak-coated Seahorse Cell Culture MicroplatesMiniplates may be stored for up to 1 week at 4°C.
  • Cell-Tak coated Cell Culture MicroplatesMiniplates Cell Culture Microplates must be allowed to warm to room temperature in the hood before cell seeding.

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2.3 Hydrate Cartridge

Basic procedures for hydrating the cartridge

An important component of the XFp HS Mini 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 Day Prior to the XFp HS Mini assay:

  • Aliquot at least 20 mL of XF Calibrant into a 50 mL conical tube. Place this in a non-CO2 37° C incubator overnight.
  • Aliquot at least 5 mL of XF Calibrant into a 15 mL conical tube. Place this in a non-CO2 37° C incubator overnight.
  • Open the Extracellular Flux Assay Kit, and remove the contents.
  • Obtain a three-pack of cartridges from the green box. Remove the foil seal from the tub(s) that will be used.
  • Place the sensor cartridge upside down next to the utility plate.
  • Separate the utility plate and Sensor Cartridge, and place the sensor cartridge upside down next to the utility plate.
  • Fill each well of the utility plate with 200 μL of sterile, tissue culture grade water.
  • Fill the moats around the outside of the wells with 400 µL per chamber.
  • Lower the sensor cartridge onto the utility plate, submerging the sensors in the water.
  • Verify the water level is high enough to keep the sensors submerged.
  • Place in a non-CO2 37° C incubator overnight. To prevent evaporation of the water, verify that the incubator is properly humidified.
  • Open the Agilent Seahorse Flux Assay Kit and remove the contents.
  • Place the Sensor Cartridge upside down next to the Utility Plate.
  • Fill each well of the Utility Plate with 1 mL of XF Calibrant.
  • Place the Hydro Booster on top of the Utility Plate.
  • Lower the Sensor Cartridge through the openings on the Hydro Booster plate, into the Utility Plate submerging the sensors in XF Calibrant.
  • Lower the sensor cartridge onto the utility plate, submerging the sensors in the water.
  • Verify the XF Calibrant level is high enough to keep the sensors submerged.
  • Place in a non-CO2 37°C incubator overnight. To prevent evaporation of the XF Calibrant, the incubator should be humidified.
  • IMPORTANT NOTE: The Hydro Booster must be removed prior to placing the Sensor Cartridge into the Agilent Seahorse Analyzer. Failure to do so may result in damage to both the Sensor Cartridge and the Analyzer. See Section 3 for further instructions.

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2.4 Design Experiment

Wave Desktop software allows you to easily create & customize assay template files to run on the Seahorse Analyzer.

What is an assay template file? Think of assay template files as an electronic copy of the experiment you designed in your lab notebook. The information entered in your assay template file is stored as a record of your experiment within the result file, which can be shared & re-run by you or other collaborators, provides structure & organization to your result data after assay completion, and offers valuable troubleshooting info when needed.

The 3 elements of an assay template file are:

  1. Group Definitions
  2. Plate Map
  3. Instrument Protocol

Group Definitions

  1. Open Wave 2.6 software
  2. Click Templates (located below Wave Home)
  3. Open the assay template called XF Real-Time ATP Rate Assay
  4. On the Group Definitions view, you will see prepopulated information for the injection strategy, pretreatments, assay media and cell type.
  5. Double-click Pretreatments and delete the Control & Experimental entries.
XF96 template selection
XF24 template selection
XFp Template Selection Icon
  1. Double-click Cell Type and delete the default entry called Cells (See Agilent Cell Analysis Publication Database).
  2. Within the Cell Type group definition, click Cell Type and Add to add a new cell type entry and enter the name of cell type you intend to analyze in your assay. It is recommended to add the seeding density to group name. For example, the C2C12 Cell Type with a seeding density of 20,000 cells per well would be named: 20k C2C12

    Repeat 3 times for each Cell Type definition.

    Note:Due to the XFe24 Analyzer's 24-well microplate format, this cell seeding density optimization protocol can be performed using 1 cell plate with 4 cell seeding densities (n=5 per group).Due to the XFe96 Analyzer’s 96-well microplate format, this cell seeding density optimization protocol can be performed using 1 cell plate with 4 cell seeding densities (n=22-24 per group).Due to the Analyzer’s 8-well miniplate format, this cell seeding density optimization protocol must be performed using two cell plates with 2 cell seeding densities per plate (n=3 per group). When designing your assay template, you can:
    • Create a new assay template for the 3rd and 4th cell seeding density groups.
    • Add 4 cell seeding density groups to one assay template and reassign the 3rd and 4th cell group to the plate map after performing the first assay with cell seeding density groups 1 and 2.
    • Rename groups in this template after performing the first assay with cell seeding density groups 1 and 2.
Group definition XFe96
Group definition XFe24
XFp group Definitions Collapsed
  1. Once finished naming your groups, click Generate Groups and Wave will automatically create your 4 unique assay groups. Notice the group name includes the cell type and seeding density for simplified plate map assignment.
    The next step is to assign groups to the plate map.

Instrument Protocol

  1. Click Instrument Protocol in the functions ribbon (under “Assay Navigation”) to view or edit the instrument protocol.

    Note: The default instrument protocol does not require modifications, however you can change the name of a protocol command, the number of measurements before/after an injection, or the length of time each measurement is performed.
    • Modifying the instrument protocol settings directly affects how data is acquired during your assay. For this example, the default instrument protocol is used (and recommended).
    • Should you need to modify the default instrument protocol, prior to performing your cell seeding density optimization assay it is recommended to review the instrument protocol section in the Wave User Guide, or if necessary, contact Agilent Cell Analysis Technical Support.
Instrument protocol XFe96
Instrument protocol XFe24
Instrument protocol XFp
  1. Last, click Run Assay in the functions ribbon (under "Assay Navigation") to add additional experimental details, save the template file, and start the cell seeding density optimization assay.
  2. Click Run Assay in the functions ribbon (under "Assay Navigation") to add additional experimental details and save the template file.
  3. Transfer the assay template file to the XFp Analyzer following steps outlined in the XFp Extracellular Flux User Guide to perform the first cell seeding density optimization assay.
  4. Transfer the assay template file to the XF HS Mini Analyzer following steps outlined in the XF HS Mini Extracellular Flux User Guide to perform the first cell seeding density optimization assay.
XFe96 review and run
XFe24 review and run
XFp review and run

3. Set up your XF assay

This section focuses on techniques performed the day of your XFp assay, including assay media preparation. Seeding non-adherent cells, and loading XFp Sensor Cartridge ports with solutions for injection.

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3.1 Prepare Cartridge & Medium

3.2 Wash Cells

3.3 Assemble Injection Solutions

3.4 Load Solutions

3.1 Prepare Cartridge & Medium

Prepare the Cartridge

  • Remove the conical tube of calibrant and assembled sensor cartridge with utility plate from the incubator.
  • Place the sensor cartridge upside down next to the utility plate.
  • Remove and discard the water from the utility plate.
  • Fill each well of the utility plate with 200 μL of the pre-warmed XF Calibrant.
  • Fill the moats around the outside of the wells with 400 μL of XF Calibrant per chamber.
  • Lower the sensor cartridge onto the utility plate, submerging the sensors in calibrant.
  • Place assembled sensor cartridge with utility plate in a non-CO2 37° C incubator for 45 – 60 minutes prior to loading the injection ports of the sensor cartridge.
  • Remove the assembled sensor cartridge with Hydro Booster and Utility plate from the incubator.
  • Place the sensor cartridge upside down next to the utility plate.
  • Remove and discard the Hydro Booster.
  • Lower the sensor cartridge onto the utility plate, submerging the sensors in calibrant.
  • Return the assembled sensor cartridge with utility plate to the non-CO2 37° C incubator until needed for loading the injection ports of the sensor cartridge.

Allow the assembled sensor cartridge with utility plate to incubate in the non-CO2 37° C incubator until needed for loading the injection ports of the sensor cartridge.

Seahorse kit

Prepare the XF Assay Medium

Seahorse assays require specific media for accurate, consistent functional measurement of metabolic activity.

Agilent provides ready-to-use, low buffered media, pre-adjusted to pH 7.4, that with compatible supplements, can streamline assay preparation and provide consistent assay conditions. View ordering information on this ready-to-use XF assay Media System or download the media selection guide.

Alternatively, researchers can formulate media with a composition that matches the assay kit being used. All compositions can be prepared using one of the Agilent Seahorse XF Media and adding different substrates/buffer as determined by the specific assay design, the example below is the Seahorse XF Real-Time ATP rate assayCell Energy Phenotype Test.

Prepare the following XF Assay Medium to use with the Seahorse XF Real-Time ATP rate assay kit.

Researchers should formulate XF assay media with a composition that matches the assay kit being used. All compositions can be prepared using one of the Agilent Seahorse XF Media and adding different substrates/buffer as determined by the specific assay design, the example below is the Seahorse XF Real-Time ATP rate assay kitCell Energy Phenotype Test.

Prepare the following XF Assay Medium to use with the Cell Energy Phenotype Test.

Agilent Reagent / Agilent Part Number Final Concentration Volume
Seahorse XF DMEM Medium, pH 7.4a, b / 103575-100 or
Seahorse XF RPMI Medium, pH 7.4a, b / 103576-100
XF Base Medium (w/out Phenol Red)a, b / 103335-100 or
XF RPMI (w/out Phenol Red)a, b / 103336-100
XF Base Medium (w/out Phenol Red)a, b / 103575-100 or
XF RPMI (w/out Phenol Red)a, b / 103576-100
- 97.0 mL 9.70 mL
Seahorse XF Glucose (1.0 M solution) / 103577-100 10 mM 10 μL 1.0 mL 100 μL
Seahorse XF Pyruvate (100 mM solution) / 103578-100 1 mM 1.0 mL 100 μL
Seahorse XF L-Glutamine (200 mM solution) / 103579-100 2 mM 1.0 mL 100 μL
aXF DMEM and RPMI Medium, pH 7.4 have a pre-adjusted pH value and do not require adjustment of pH upon addition of XF supplements. See method below for preparation.Seahorse XF DMEM Medium pH 7.4 and RPMI Medium, pH 7.4 are not compatible with XF24 Analyzers.
bPreparation using alternative types of XF mediaPreparation using alternative types of XF mediaPreparation using alternative types of XF mediaPreparation using alternative types of XF mediaPreparation using alternative types of XF media.

Basic procedures for preparing XF DMEM Medium pH 7.4 or XF RPMI Medium pH 7.4

Basic procedures for preparing XF Base Medium (w/o Phenol Red) or XF RPMI (w/o Phenol Red)

Equipment Required:

  • 37°C water bath
  • Calibrated pH meter
  • Stir plate
  • Sterile filter bottles (0.22 μm filter) and cap
  • 1.0 N NaOH solution

Agilent Seahorse XF DMEM Medium pH 7.4 and XF RPMI Medium pH 7.4 are designed to provide:

  • Convenience: No adjustment of final pH is required when used as recommended with Agilent Seahorse XF Supplements.
  • Consistency: Low concentrations of HEPES buffer (5 mM, DMEM; 1 mM, RPMI) provide more consistent XF data.
  • Quantitation: Using assay medium with a fixed buffer capacity allows for quantitative measurement of proton efflux rate (PER).
  1. Warm appropriate volume of XF DMEM Medium pH7.4 or XF RPMI Medium pH 7.4 to 37°C in a sterile bottle. In general, 100 mL is sufficient for one plate.
  2. Warm appropriate volume of XF Base Medium (w/o Phenol Red) or XF RPMI (w/o Phenol red) to 37°C in a sterile bottle. In general, 100 mL is sufficient for one XF24 plate.
  3. Add appropriate volumes of Seahorse XF supplements (XF Glucose solution, XF Pyruvate solution and XF L-Glutamine solution) indicated in the table above.
  4. Adjust pH value of the medium to 7.4 using 1 N NaOH. Note: pH value will change quickly upon addition of NaOH, use small volumes and add slowly to adjust pH value.
  5. Sterilize assay medium with a 0.2 μm filter.
  6. Incubate the final XF Assay Medium at 37°C until ready for use

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3.2 Wash Cells

Basic procedure for washing adherent cells

For adherent cells seeded at least one day prior to the XFp HS Mini assay:

  • Retrieve the cell culture miniplate from the CO2 incubator.
  • View the cells under the microscope to:
    1. Confirm cell health, morphology, seeding uniformity and purity (no contamination).
    2. Ensure cells are adhered, with a consistent monolayer.
    3. Make sure there are no cells in the background correction wells.
  • Wash adherent cells with complete assay medium:one time with XF Real-Time ATP Rate Assay Media:
    1. 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.
    2. Gently add 200 μL of assay medium.
    3. Place the plate in a 37°C incubator without CO2 for one hour prior to the assay.
    1. Remove all but 50 μL of the culture medium from each well. The small amount of medium is left to keep the cells from drying out.
    2. Gently add 1mL of assay medium.
    3. Place the plate in a 37°C incubator without CO2 for one hour prior to the assay.
    4. Repeat step b, removing all but 50 μL (as in step a).
    5. Add 450 μL assay medium (to a total volume of 500 μL) for a 24 well platform instrument.
    6. Just prior to starting the assay, wash the cells again with XF Real-Time ATP Rate Assay Media: remove all media but 50 μL and add fresh media to a final volume 500 μL. Inspect the cells under the microscope to ensure that cells were not disturbed or washed away.
  • Just prior to starting the assay, wash the cells again with XF Real-Time ATP Rate Assay Media: remove all media but 20 μL and add fresh media to a final volume 180 μL. Inspect the cells under the microscope to ensure that cells were not disturbed or washed away.
  • Observe the assay wells under the microscope to ensure that cells were not washed away.
  • Place the plate in a 37° C incubator without CO2 for one hour prior to the assay.

Basic procedures for seeding suspension cells

The optimal cell density for suspension cells varies depending on the cell size. In general, optimal cell seeding density should result in cell distribution in the well as a monolayer at 70-90% confluency. It is strongly encouraged to examine cell distribution under a microscope to look for (1) adequate space between cells to ensure all cells contact the coated surface evenly and (2) ensure minimal cell clusters. Seeding an excess number of cells above the optimal density or if the cells cluster together can result in poor cell adhesion and cause inaccurate rate measurements.

Cell image A
Cell image B
  • For one Seahorse Cell Culture Microplate, transfer an appropriate volume of cell suspension from the growth vessel to a conical tube.
  • To calculate the total number of cells needed, multiply the desired number of cells per well times 100 wells for the Seahorse XFe96. (For example, 150,000 cells per well × 100 wells = 1.5 × 107 cells).
  • To calculate the total number of cells needed, multiply the desired number of cells per well times 10 wells for the Seahorse . (For example, 150,000 cells per well × 10 wells = 1.5 × 106 cells).
  • Centrifuge cells at room temperature at 200 × g for 5 minutes.
  • While cells are being centrifuged, pipette 50 μL assay medium into background/control wells of the prewarmed PDL-coated Seahorse XF96 Cell Culture Microplates or Cell-Tak-coated Seahorse XF96 Cell Culture Plate.
  • While cells are being centrifuged, pipette 50 μL assay medium into background/correction wells (A and H) of the prewarmed PDL-coated Seahorse XFp Cell Culture Microplates or Cell-Tak-coated Seahorse XFp Cell Culture Plate.
  • Remove supernatant from the centrifuged conical tube.
  • Resuspend cells in warmed assay medium to the desired concentration of cells per well in 50 μL of assay medium. (For example, 1.5 × 105 cells per well is desired, resuspend cells in a volume that results in 1.5 × 105 cell/50 μL or 3.0 × 106 cells/mL).
  • Change centrifuge settings to zero braking.
  • Transfer the cell suspension to a sterile tissue culture reservoir, or pipette from the conical tube.
  • Pipette 50 μL of the cell suspension along the side of each well, except for background/control wells. It is recommended to use a multichannel pipette.
  • Place the miniplate(s) in an XFp carrier tray and centrifuge at 300 x g for 1 min with no brake. The carriers are designed to hold up to 3 miniplates, and fit standard centrifuge microplate adapters. Ensure that the centrifuge rotor is balanced appropriately.
  • After centrifugation, visually confirm adherence of the cells to the well bottom.
  • Centrifuge the cells at 200 × g (zero braking) for 1 minute. Ensure that the centrifuge is properly balanced.
  • Taking care not to disturb the cells on the bottom, gently add 130 μL assay medium to each well to the desired initial assay volume (for 180 μL starting assay volume).
  • Add sterile water or PBS to the moat around the cell culture wells, 100 μL per chamber. 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).
  • Transfer plates to a 37° C incubator not supplemented with CO2 for 25–30 minutes to ensure that the cells have completely attached. Visually confirm that most of the cells are stably adhered to the culture surface.
  • Slowly and gently, add 130 μL warm assay medium along the side of each well. Take care to avoid disturbing the cells.
  • Observe the cells under the microscope to check that cells are not detached.
  • Return the cell plate to the incubator for 15–25 minutes.
  • After 15–25 minutes, the cell plates are ready for your assay. Total time following centrifugation should be no greater than 1 hour for best results.
  • For one Seahorse XF24 Cell Culture Microplate, transfer an appropriate volume of cell suspension from the growth vessel to a conical tube.
  • To calculate the total number of cells needed, multiply the desired number of cells per well times 25 wells for the Seahorse XF24. (For example, 150,000 cells per well × 25 wells = 3.75 × 106 cells).
  • Centrifuge cells at room temperature at 200 × g for 5 minutes.
  • While cells are being centrifuged, pipette 100 μL assay medium into background/control wells of the room-temperature Cell-Tak-coated Seahorse XF24 Cell Culture Plate.
  • Remove supernatant from the centrifuged conical tube.
  • Resuspend cells in warmed assay medium to the desired concentration of cells per well in 100 uL of assay medium. (For example, 1.5 × 105 cells per well is desired, resuspend cells in a volume that results in 1.5 × 105 cell/100 μL or 1.5 × 106 cells/mL).
  • Change centrifuge settings to zero braking.
  • Transfer the cell suspension to a sterile tissue culture reservoir, or pipette from the conical tube.
  • Pipette 100 μL of the cell suspension along the side of each well, except for background/control wells. Agilent recommends using a multichannel pipette.
  • Centrifuge the cells at 200 × g (zero braking) for 1 minute. Ensure that the centrifuge is properly balanced. For XFp Analyzer users, Agilent recommends using the Agilent Seahorse XFp Carrier Tray to centrifuge the Seahorse XFp Cell Culture Miniplates. For more details, refer to the Basic Procedure: Seeding Suspension Cells in XFp Cell Culture Miniplates.
  • Transfer plates to a 37° C incubator not supplemented with CO2 for 25–30 minutes to ensure that the cells have completely attached. Visually confirm that most of the cells are stably adhered to the culture surface.
  • Slowly and gently, add 400 μL warm assay medium along the side of each well. Take care to avoid disturbing the cells.
  • Observe the cells under the microscope to check that cells are not detached.
  • Return the cell plate to the incubator for 15–25 minutes.
  • After 15–25 minutes, the cell plates are ready for your assay. Total time following centrifugation should be no greater than 1 hour for best results.

Related Support Material

Reference Material

3.3 Assemble Injection Solutions

A key feature of the Agilent Seahorse Analyzer is its ability to inject reagents during the assay and see results in real time. This is accomplished by dispensing solutions that have been loaded into injector ports within the cartridge prior to placement in the instrument.

If performing initial cell characterization of cell density and/or FCCP titrations using the Cell Energy Phenotype Assay, prepare injection solution as described in the tables below.

If performing initial cell characterization of cell density using the Seahorse XFp Real-Time ATP rate assay, prepare injection solution as described in the tables below.

If performing initial cell characterization of cell density using the Seahorse XF Real-Time ATP rate assay, prepare injection solution as described in the tables below.

Cell Density Titration Assay Solution Assembly

FCCP Concentration Titration Assay Solution Assembly

  • Remove one pouch from the Seahorse Seahorse XF Real-Time ATP rate assay Kit box, and remove both tubes (Oligo and Rotenone + Antimycin A).
  • Remove one pouch from the Seahorse XF Cell Energy Phenotype Test Kit box, and remove both tubes (Oligo and FCCP).
  • Using a pipette, resuspend the contents of each tube with prepared assay medium using the volumes described in the table below. Place a cap on the tube, and vortex for 1 minute to solubilize the compounds.
Resuspension volumes for the Seahorse XF Real-Time ATP rate assay Kit
Compound Volume of XF Assay Media Resulting Stock Concentration
Oligomycin 420 µl 168 µl 150 µM 75 µM
Rotenone + Antimycin A 540 µl 216 µl 50 µM 25 µM
Resuspension volumes for the XF Cell Energy Phenotype Test Kit
XF Cell Energy Phenotype Test Component Volume of XF assay media (μL) Resulting Stock Concentration (μM)
Oligomycin 630 100
FCCP 720 100
  • Prepare 3.0 mL of each injection solution by combining the appropriate volumes of XF Assay Media and stock oligomycin and stock rotenone/antimycin A as described in the table below.
  • Using a 15 mL conical tube, prepare 3.0mL of the injection solution by combining the appropriate volumes of XF Assay Media, stock oligomycin and stock FCCP as described in the table below.
  • Prepare 300 µL of each injection solution by combining the appropriate volumes of XF Assay Media and stock oligomycin and stock rotenone/antimycin A as described in the table below.
Dilution volumes for XF Real Time ATP Rate Assay Kit - Cell Characterization
Port & Compound Stock Volume XF Assay Media Volume 10X [Port] [Final Well]
Port A Oligomycin 300 µl 60 µl 2700 µl 240 µl 15 µM 1.5 µM
Port B Rotenone + Antimycin A 300 µl 60 µl 2700 µl 240 µl 5 µM 0.5 µM
Dilution volumes for the XF Cell Energy Phenotype Test Kit - Cell Seeding Density Titration with XFe24/XF24
Final FCCP concentration in well Volume of assay media (μL) Volume of Stock Oligomycin (μM) Volume of Stock FCCP (μL) 10X Final Oligo (Port) Concentration (μM) 10X Final FCCP (Port) Concentration (μM)
0.25 875 100 25 10 2.5
0.5 850 100 50 10 5.0
1.0 800 100 100 10 10
2.0 700 100 200 10 20

If performing a different type of XFp HS Mini assay, consult the appropriate XFp HS MiniKit User Guide for appropriate injection solution preparation instructions.

Sours: https://www.agilent.com/en/product/cell-analysis/how-to-run-an-assay

An Optimized Protocol to Analyze Glycolysis and Mitochondrial Respiration in Lymphocytes

References

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Sours: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5226256/
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Measuring bioenergetics in T cells using a Seahorse Extracellular Flux Analyzer

Critical parameters and troubleshooting

All cell preparations should be done as quickly as possible, and all cells must be kept on ice while working with one cell population at a time. Accurate cell counting is critical for adequate data comparison between groups. If T cells are isolated using a cell sorter, they should be re-counted after sorting, as during sorting, cells may die, thus sorter counts are not sufficiently accurate. In addition, a fixed number of cells should be seeded in each well, and it is recommended to have a minimum of 4 wells per group. If one would like to compare data between different plates/runs, a common control, such as naïve T cells, or a group of cells treated in the same condition as previous runs could possibly be included with each plate to normalize the data between the different runs.

Before seeding the cells, ensure that the cell plate is at room temperature. After plating the cells and the addition of XF media, wells should be checked under a microscope to confirm an intact monolayer of cells in all wells. In case only part of the plate is used, it is critical to fill all injection ports (of the ports that will be injected, for Basic Protocol 1 that is ports A–C). This will avoid inconsistent injection of drugs into wells due to unbalanced pressure. It is recommended to fill all unused wells in the cell plate with XF media as these can function as additional background wells. In order to eliminate CO2 perturbations and to reduce potential measurement variation during the run, it is important to incubate the cell plate in a 37°C non-CO2 incubator for at least 30 minutes but not more than 60 minutes before putting it in the EFA. Table 1 lists some potential problems and suggestions.

Table 1

Troubleshooting Guide for Measuring Bioenergetics with an Extracellular Flux Analyzer

ProblemComments and Suggestions
Poor basal signal
 - Low cell numberIncrease the number of cells per well.
 - Low cell viabilityKeep cells on ice for all steps before plating and/or reduce processing time.
High variation between replicates
 - Uneven cell number platedMake sure the correct volume of cells is added in each well of the cell plate, and that the volume of cells is maximal 100 μl.
 - A certain probe does not detect signals properlyMake sure all probes are hydrated for 4 – 24 hours with sufficient volume of Calibrant XF, and use cartridges before the expiration date.
 - Cell monolayer was disturbed during XF media additionMake sure the cell plate is PDL-coated and the plate has been spun down after adding the cells. Using too many cells, as well as having
substantial numbers of dead cells in the well, results in multiple cell layers, and will increase disturbance of the cells after injection.
High variation between repeated measurements within a loop
 - Decline in basal measurementsMake sure to keep the cell plate in a 37°C non-CO2 incubator for 30–60 minutes before putting the cells in the machine.
 - Decline in measurements after drug injectionsThis is sometimes, but not exclusively, seen after FCCP injection. The drug concentration may not be optimal. Titration of the drug might eliminate this problem, however, other biological factors, independent of drug dose, could also contribute to a drop.
 - Cell monolayer was disturbed after injectionMake sure the cell plate is PDL-coated and the plate is spun down after plating the cells. Using too many cells, as well as having substantial numbers of dead cells in the well, results in multiple cell layers, and will increase disturbance of the cells after injection.
Minimal or unexpected changes in OCR after drug injection
 - No drug is injectedMake sure all drugs are added directly into the correct ports in the right orientation without contaminating other ports.
 - Inconsistent injection of drugs between wellsFill all injection ports that will be injected with drugs of interest. Make sure all injection ports that will be injected are filled with the proper volume to ensure balanced injection.
 - Concentration of drug is too lowTitration of drug concentration may be required.
 - Incorrect orientation of cartridge or cell plate during plating or runningThe drugs will be injected in a wrong order if the orientation of the cartridge is incorrect. Ensure correct orientation (see user's manual of the EFA) of the cell plate and the cartridge, both during plating/drug loading and during the run.

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Anticipated results

Measurement of T cell bioenergetics using the Seahorse EFA will vary depending on the type and doses of drugs, measuring time, cell numbers and the pre-treatment of cells. Figure 2 demonstrates typical OCR curves of naïve, effector and memory CD8+ T cells from wild-type mice infected with Listeria monocytogenes using drugs described in Basic Protocol 1. OCR should decrease after the addition of oligomycin, and increase after FCCP injection. The extent of the increase in OCR after FCCP depends on the subset of T cells (van der Windt et al., 2012). SRC can be calculated as the ratio of maximum OCR after FCCP injection/basal OCR. Figure 3 shows an immediate increase in ECAR in T cells upon PMA/ionomycin injection, indicating the engagement of glycolysis after T cell stimulation (van der Windt et al., 2013).

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Figure 2

CD8+ T cells were isolated from spleens and lymph nodes harvested from naïve (for naïve T cells: TN) and Listeria monocytogenes infected mice (effector cells : TE, and memory cells: TM). O2 consumption rates (OCR) of CD8+ T cells were measured in real-time under basal conditions and in response to indicated mitochondrial inhibitors. Notice that TM cells have considerable spare respiratory capacity (SRC). Figure was originally published in Immunity (van der Windt et al., 2012).

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Figure 3

OT-I cells were activated with OVA-peptide and IL-2 for 3 days and subsequently cultured in the presence of IL-15 for 3 days to generate IL-15 memory (TM) cells. Extracellular acidification rates of naive (TN) and IL-15 TM cells, in resting conditions, and after stimulation with PMA/ionomycin or control (injected after 5 measurements). Figure was originally published in PNAS (van der Windt et al., 2013).

Time considerations

The preparation of the cells for bioenergetic measurement depends strongly on the number of groups. Typically this process takes 30–120 min, including the harvesting and counting of cells, the adjustment to the desired cell concentrations, the plating of cells and the addition of XF media. Plating the drugs into the ports of the cartridge usually requires 20 minutes, but this also depends on the number and combinations of drugs used in the experiment. As such, the time considerations are rough guidelines and it is recommended that an investigator becomes familiar with the technique before assessing many groups on a plate. In addition, the waiting time after mixing and before measurement is crucial for the XFe24 EFA, but it is optional for the XFe96 model. Finally, the Seahorse EFA running time for a standard mitochondrial stress test (as described in Basic Protocol 1) takes 160 minutes, including the time for the cartridge calibration and equilibration steps.

Sours: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4864360/
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