How to run an assay

Procedures, techniques & resources for a successful Assay

Your XF Instrument Type

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 Cell Energy Phenotype Test Real Time ATP Rate Assay 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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+ Gather Assay Materials

+ 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 XFp assay, including guidance for choosing cell seeding densities, techniques for seeding adherent cells on XFp tissue culture plates and hydrating XFp cartridges.

Select a workflow step in the image below to display help content.

Cell Seeding Densities

Seeding Cells

Hydrate Cartridge

Design Experiment

Choose Cell Seeding Densities

Basic procedures for choosing cell seeding densities

Training Webinar: Prepare for your XF Assay

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 XF Real-Time ATP rate assay Test Kit The Seahorse XFp Real-Time ATP rate assay Test Kit The Seahorse Cell Energy Phenotype 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. 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.

Seeding Cells

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

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

Agilent Seahorse XFp Assays are performed in an Agilent Seahorse 96-well 24-well 8-well XFp Cell Culture Microplate Miniplate 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 instrument are recommended for initial assays.

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

Seeding for Cell Density Titration Assay

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

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:

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 approxima- tion 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.

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

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.
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 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.
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.
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.
¹Lundholt 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 assay)

Prepare Cell-Tak coated Cell Culture plate(s) Miniplate(s) as described below:

The optimal Cell-Tak solution concentration for Agilent Seahorse Cell Culture Microplate Miniplate is 22.4 μg/mL.
Prepare 2.5 mL 1.5 mL 0.25 mL of this solution per plate for each assay. Refer to the Manufacturer's protocol to prepare this solution.
Apply 25 50 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 Microplates Miniplates may be stored for up to 1 week at 4°C.
Cell-Tak coated Cell Culture Microplates Miniplates Cell Culture Microplates must be allowed to warm to room temperature in the hood before cell seeding.

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Hydrate Cartridge

Basic procedures for hydrating the cartridge

An important component of the XFp 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 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.
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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Design Experiment

Wave Desktop software is used for designing XF assays for the Seahorse XFe and XFp Analyzers. Use the resources below to learn more about using Wave Desktop software for assay design, including the elements of an assay template, modifying instrument protocol for custom assays, managing template files, and using the prepopulated catalog.

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:

Group Definitions
Plate Map
Instrument Protocol

Group Definitions

Open Wave 2.6 software
Click Templates (located below Wave Home)
Open the assay template called XF Real-Time ATP Rate Assay
On the Group Definitions view, you will see prepopulated information for the injection strategy, pretreatments, assay media and cell type.
Double-click Pretreatments and delete the Control & Experimental entries.
Double-click Cell Type and delete the default entry called Cells (See Cell Reference Database).
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).

Note: 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).
Note: Due to the XFp 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.

Generate Groups

The next step is to assign groups to the plate map.

Plate Map

Click Plate Map in the functions ribbon (under "Assay Navigation").
Assign the first cell seeding density group to the plate map. To assign a group to the plate map, first click the group name in the group list, then:
- Click the column header (i.e. 1, 2, 3, etc.) or the row header (i.e. A, B, C, etc.)
- Drag & drop an area of wells on the plate map using your mouse left-click.
- Click individual wells on the plate map (not recommended for this assay template).
Repeat for the next cell seeding density group. The suggested plate map layout is pictured above/below.

Instrument Protocol

Click Instrument Protocol in the functions ribbon (under “Assay Navigation”) to view or edit the instrument protocol.
Note: The default instrument protocol is 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.
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.

Click Run Assay in the functions ribbon (under "Assay Navigation") to add additional experimental details and save the template file.
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.

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+ 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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Prep Cartridge/Media

Wash Cells

Assemble Solutions

Load Solutions

Prepare Cartridge and Assay Medium

Prepare the Cartridge

Training Webinar
Set up and run your XF Assay

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.

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 Cell Energy Phenotype Test Seahorse XF Real-Time ATP rate assay.

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 Cell Energy Phenotype Test Seahorse XF Real-Time ATP rate assay kit.

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

Agilent Reagent / Agilent Part Number	Final Concentration	Volume
Seahorse XF DMEM Medium, pH 7.4^{a, b} / 103575-100 or Seahorse XF RPMI Medium, pH 7.4^{a, 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	-	97.0 mL 9.70 mL
Seahorse XF Glucose (1.0 M solution) / 103577-100	10 mM	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
^a XF 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. ^a Seahorse XF DMEM Medium pH 7.4 and RPMI Medium, pH 7.4 are not compatible with XF24 Analyzers. ^b Preparation using alternative types of XF media. ^b Preparation using alternative types of XF media. ^b Preparation using alternative types of XF media. ^b Preparation using alternative types of XF media. ^b Preparation using alternative types of XF media.

Basic procedures for preparing XF Base Medium (w/o Phenol Red) or XF RPMI (w/o Phenol Red) Basic procedures for preparing XF DMEM Medium pH 7.4 or XF RPMI Medium pH 7.4

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).

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.
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.
Add appropriate volumes of Seahorse XF supplements (XF Glucose solution, XF Pyruvate solution and XF L-Glutamine solution) indicated in the table above.
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.
Sterilize assay medium with a 0.2 μm filter.
Incubate the final XF Assay Medium at 37°C until ready for use

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Wash Cells

Basic procedure for washing adherent cells

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

Retrieve the cell culture miniplate from the CO₂ incubator.
View the cells under the microscope to:

Confirm cell health, morphology, seeding uniformity and purity (no contamination).
Ensure cells are adhered, with a consistent monolayer.
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:

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.
Gently add 200 μL of assay medium.
Place the plate in a 37°C incubator without CO₂ for one hour prior to the assay.

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.

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.
Gently add 1mL of assay medium, then remove the same amount.
Repeat step b, removing all but 50 μL (as in step a).
Add 450 μL assay medium (to a total volume of 500 μL) for a 24 well platform instrument.

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.
Gently add 1mL of assay medium.
Place the plate in a 37°C incubator without CO₂ for one hour prior to the assay.
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.

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

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 XFp. (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 room-temperature 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 room-temperature 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.

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Assemble 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 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 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

Remove one pouch from the Seahorse Seahorse XF Real-Time ATP rate assay Kit box, and remove both tubes (Oligo and Rotenone + Antimycin A).

Resuspension volumes for the Seahorse XF Real-Time ATP rate assay Kit
Compound	Volume of XF Assay Media	Resulting Stock Concentration
Oligomycin	420 µl	150 µM
Rotenone + Antimycin A	540 µl	50 µM

Remove one pouch from the Seahorse XFp Real-Time ATP rate assay box, and remove both tubes (Oligo and Rotenone/Antimycin A).

Resuspension volumes for the Seahorse XF Real-Time ATP rate assay Kit
Compound	Volume of XF Assay Media	Resulting Stock Concentration
Oligomycin	168 µl	75 µM
Rotenone + Antimycin A	216 µl	25 µM

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.

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

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.

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	2700 µl	15 µM	1.5 µM
Port B Rotenone + Antimycin A	300 µl	2700 µl	5 µM	0.5 µM

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	60 µl	240 µl	15 µM	1.5 µM
Port B Rotenone + Antimycin A	60 µl	240 µl	5 µM	0.5 µM

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

+ Related Support Material

Load Injection Solutions

If performing initial cell characterization (Cell Density and FCCP Concentration Titration Assay) using the Cell Energy Phenotype Assay, follow the instructions and table below to load the cartridge injection ports. If performing initial cell characterization (Cell Density Assay) using the Seahorse XF Real-Time ATP rate assay, follow the instructions and table below to load the cartridge injection ports.

Note that for these assay designs, only the A and B injection ports will be used.

Sensor Cartridge Injection Port Layout

XF24 Sensor Cartridge Injection Port Layout

Remove the hydrated cartridge from the non-CO₂ incubator.
Orient the Agilent Seahorse XF Assay Cartridge. Place row labels (lettered A-H) to the left. The triangular notch will be in the bottom left-hand corner.
Orient the Agilent Seahorse XF Assay Cartridge. Place row labels (lettered A-D) to the left. The triangular notch will be in the bottom left-hand corner.
Orient the Agilent Seahorse XFp Assay Cartridge. Place well labels (lettered A-H) to the left. The triangular notch will be in the bottom left-hand corner.
XFp Sensor Cartridge Injection Port Layout
Place the A/D loading guide flat on top of the assay cartridge. Orient the loading guide so the letter 'A' is located in the upper left-hand corner. Use your fingertips to hold the outside edges of the loading guide to stabilize during loading so pipette tips do not dislodge the loading guide.
Using a 10-100 μL multichannel pipette, make sure the tips are securely fitted onto the pipette. Position the pipette tips (filled with your compounds for injection) into the desired column in the loading guide, and orient the tips at a very slight angle. Insert the tips as far as they will go without resistance into the holes and dispense the compound. Do not force the tips completely into the holes.
Using a 100 or 200 μL (multichannel) pipette, make sure the tip(s) are securely fitted onto the pipette. Position the pipette tip(s) (filled with your compounds for injection) into the desired port, and orient the tips at a very slight angle (<5°). Do not force the tips completely into the holes.
Hold the tips at a 45° angle. Place the tips halfway into the injection ports with the bevel of the tip against the opposite wall of the injection port.
Do NOT insert the tips completely to the bottom of the injection ports as this may cause compound leakage through the port.
Gently dispense 20 μL of the appropriate injection solution into the ports according to plate/group layouts shown below. Withdraw the tips from the ports carefully stabilizing the loading guide throughout the procedure. Avoid creating air bubbles. Do not tap any portion of the cartridge in an attempt to alleviate air bubbles. This may cause solutions to leak from the injection port.
Gently dispense 55 μL of the appropriate injection solution into the ports according to plate/group layouts shown below, depending on which assay is being performed. Withdraw the tips from the ports carefully. Avoid creating air bubbles, but do not tap any portion of the cartridge in an attempt to alleviate air bubbles. This may cause solutions to leak from the injection port.
Gently dispense 20 μL of the appropriate injection solution into the ports according to plate/group layout shown below. Withdraw the tip(s) from the ports carefully, stabilizing the cartridge throughout the procedure. Avoid creating air bubbles. Do not tap any portion of the cartridge in an attempt to alleviate air bubbles. This may cause solutions to leak from the injection port.
Remove the A/D port loading guide, and replace with the B/C port loading guide, with the ‘B’ in the upper left corner. Repeat steps above to load port B, using 22 µl of injection solution.
Repeat steps above to load port B, using 62 µl of injection solution.
Repeat steps above to load port B, using 22 µl of injection solution.
Visually inspect the injection ports for even loading. The liquid should be in the port, make sure there are no residual drops on top of the cartridge.

Injection Port Loading for Cell Density Titration Assay

Injection Ports and volumes for XF Real Time ATP Rate Assay Kit Cell Characterization
Port & Compound	Volume	10X [Port]	[Final Well]
Port A Oligomycin	55 µl	15 µM	1.5 µM
Port B Rotenone + Antimycin A	62 µl	5 µM	0.5 µM

Final concentration in well (μM)	FCCP Group	Wells	Port/Volume (μL)
Oligo / FCCP 1.0 / 1.0	1.0 μM	A1-D6 (all)	A / 55

Injection Port Loading for FCCP Concentration Titration Assay

Injection Ports and volumes for XF Real Time ATP Rate Assay Kit Cell Characterization
Port & Compound	Volume	10X [Port]	[Final Well]
Port A Oligomycin	20 µl	15 µM	1.5 µM
Port B Rotenone + Antimycin A	22 µl	5 µM	0.5 µM

If performing a different type of XF assay, consult the appropriate XF Kit User Guide and the instructions below for appropriate loading methods for more than one injection solution.

Orient the Agilent Seahorse XF Assay Cartridge. Place row labels (lettered A-H) to the left. The triangular notch will be in the bottom left-hand corner.
Orient the Agilent Seahorse XF Assay Cartridge. Place row labels (lettered A-D) to the left. The triangular notch will be in the bottom left-hand corner.
Orient the Agilent Seahorse XF Assay Cartridge. Place row labels (lettered A-D) to the left. The triangular notch will be in the bottom left-hand corner.
Place the A/D loading guide flat on top of the assay cartridge. Orient the loading guide so the letter 'A' is located in the upper left-hand corner. Use your fingertips to hold the outside edges of the loading guide to stabilize during loading so pipette tips do not dislodge the loading guide.
Using a 10-100μl multichannel pipette, make sure the tips are securely fitted onto the pipette. Position the pipette tips (filled with your compounds for injection) into the desired column in the loading guide, and orient the tips at a very slight angle. Insert the tips as far as they will go without resistance into the holes and dispense the compound. Do not force the tips completely into the holes.
Using a multichannel pipette, make sure the tips are securely fitted onto the pipette.
Hold the tips at a 45° angle. Place the tips halfway into the injection ports with the bevel of the tip against the opposite wall of the injection port.
Do NOT insert the tips completely to the bottom of the injection ports as this may cause compound leakage through the port.
Dispense the compounds into the ports gently. Withdraw the tips from the ports carefully , stabilizing the loading guide throughout the procedure.
Avoid creating air bubbles, but do not tap any portion of the cartridge in an attempt to alleviate air bubbles. This may cause compound leakage from the injection port.
Switch to the B/C loading guide. Orient with the letter 'B' in the upper left-hand corner. Repeat loading procedure outlined in steps 3-5 for 'B', 'C' and 'D' injection ports, using the appropriate loading guides. Remove and discard loading guide(s).
Repeat loading procedure outlined in steps above for 'B', 'C' and 'D' injection ports.
Visually inspect the injection ports for even loading. The liquid should be in the port, make sure there are no residual drops on top of the cartridge.

General Information and Guidelines for Injections

The recommended injection volume is 20-30 μL.
The recommended injection volume is 50-100 μL.
Recommended Injection Solution Volumes for 10X dilution upon injection, starting with a microplate well volume of 180 μL assay medium:
- Port A: 20 μL
- Port B: 22 μL
- Port C: 25 μL
- Port D: 28 μL
Recommended Injection Solution Volumes for 10X dilution upon injection, starting with a microplate well volume of 500 μL assay medium:
- Port A: 55 μL
- Port B: 62 μL
- Port C: 69 μL
- Port D: 75 μL
The composition, sequence and number of ports utilized will depend on your assay design.
Automated pipettes are not recommended for cartridge loading, as they may lead to injection solutions leaking through the port orifice.

+ Related Support Material

+ Run your XF Assay

This section focuses on performing initial cell characterization using the XF Cell Energy Phenotype Test on your Analyzer.

This section focuses on performing initial cell characterization using the XF Real-Time ATP Rate Assay on your Analyzer.

Important – Before you start your XF Assay

Visually inspect the injection ports for even loading. The liquid should be in the port, make sure there are no residual drops on the top of the sensor cartridge.
View cells under a microscope to:
- Confirm cell health, morphology, seeding uniformity and purity (no contamination).
- For adherent cells, ensure cells are adhered with a consistent monolayer and were not washed away during washing step.
- For suspension cells, ensure cells are stably adhered following centrifugation, washing and incubation.
- Ensure your background wells do not contain cells.

Procedure to Run your Assay

Once all required injection ports are filled, transfer the cartridge and utility plate to the Analyzer and begin cartridge calibration.
Important! Before starting calibration, ensure:
- The sensor cartridge fits properly on the Utility plate.
- The lid is removed from the sensor cartridge.
- Proper orientation (direction) of the sensor cartridge on the Utility plate.
When the cartridge calibration is complete, follow the software prompts to exchange the utility plate for the cell culture plate and initiate the XF assay.
Important! Before loading the cell plate, ensure:
- The lid is removed the Cell Plate.
- Proper orientation (direction) of the Cell Plate on the tray.
When your assay is complete eject the sensor cartridge & cell culture plate, set aside for later analysis if necessary (example - cell count normalization).
Save or transfer the XFd result file to a shared network drive or USB drive and open using Wave Desktop software on your PC for analysis of assay data.

Go to the and ensure the XFe Analyzer is powered ON and connected to the XFe Controller (computer). You can verify the instrument connection status in the widget panel in the lower-left corner of Wave Controller software.
Double-click to open the desired assay template file from the Templates view. If your assay template is not displayed on the Templates view, transfer the template using a shared network drive or USB flash drive.
After reviewing the group definitions, plate map layout and instrument protocol, click Start Run.
After you enter the save location for your result file (following completion of the assay), the tray door on the XFe Analyzer will open.
Important! Before starting calibration, ensure:
- The sensor cartridge fits properly on the Utility plate.
- The lid is removed from the sensor cartridge.
- Proper orientation (direction) of the sensor cartridge on the Utility plate.
Place the sensor cartridge (hydrated and loaded with compounds) and Utility plate onto the tray when prompted.
Press I'm Ready to initiate sensor cartridge calibration.
Time to complete calibration is approximately 10-20 minutes (for assays at 37°C). For XF assays performed at temperatures other 37°C an additional 30 minutes of pre-calibration time will be added to ensure accurate data acquisition.

Once sensor cartridge calibration is complete, Wave Controller will display the Load Cell Plate dialog.
Click Open Tray to eject the Utility plate and load the Cell Plate on the tray. The sensor cartridge remains inside the XFe Analyzer for this step.
Important! Before loading the cell plate, ensure:
- The lid is removed the Cell Plate.
- Proper orientation (direction) of the Cell Plate on the tray.
After placing the Cell Plate on the tray, click Load Cell Plate to initiate equilibration.
After completing equilibration, the assay will automatically begin acquiring baseline measurements (as outlined in your instrument protocol).
Once the final measurement command in the instrument protocol is completed, Wave Controller software will display the Unload Sensor Cartridge dialog.
Click Eject when ready to eject the sensor cartridge and cell plate. Set aside for later analysis if necessary (example - cell count normalization).
After removing the sensor cartridge and cell plate, the Assay Complete dialog will appear.
Click View Results to immediately open your assay result file, or click Wave Home to return to the Templates view and begin another XFe assay.
Save or transfer the assay result file to a shared network drive or USB drive and open using Wave Desktop software on your PC for data analysis.

See Chapter 2 of the Wave User Guide for complete details of XFe Analyzer assay operation, including how to add/remove measurements during an assay, environmental conditions for XFe assays at temperatures other than 37°C, and more.

On the XFp Analyzer home screen, touch Start to display the list of available assay templates.
If the desired template is not available under the Local tab, open or transfer the template file using a shared network drive or USB flash drive.
Touch to open the assay template to review the template design:
1. Group definitions- Touch the group name to display the injection strategy, pretreatments, and cell type information for the selected group. XFp Analyzer software does not allow modifications to group definitions, Wave Desktop software must be used.
2. Plate Map - Change a well assigned to a group, touch the group name first, then touch the well on the plate map.
Touch the right arrow (lower-right corner) to review or edit the instrument protocol.
1. To add or remove measurement cycles from the instrument protocol, first touch the protocol step, then use the plus/minus buttons to adjust measurement cycles.
Touch the right arrow again (lower-right corner) to display the review & run screen. Before you press Start Run, you can:
1. Press Edit next to Assay Name to customize the name of your assay result file.
2. Press Edit next to Notes to add custom notes related to your assay.
3. Press Edit next to Email Notification to notify recipients for user-interaction (example – replace utility plate with the cell plate), and to automatically send the assay result file following completion of the assay. This feature requires an active internet connection on the XFp Analyzer.
When ready to begin the XF assay, click Start Assay.
Place the sensor cartridge (hydrated & loaded with compounds) and utility plate onto the tray when prompted.
Important! Before starting calibration, ensure:
- The sensor cartridge fits properly on the Utility plate.
- The lid is removed from the sensor cartridge.
- Proper orientation (direction) of the sensor cartridge on the Utility plate.
Once calibration is complete the XFp Analyzer tray will open and present the utility plate. Remove the utility plate and load the cell plate on the tray.
Important! Before loading the cell plate, ensure:
- The lid is removed the Cell Plate.
- Proper orientation (direction) of the Cell Plate on the tray.
After placing the Cell Plate on the tray, touch Continue to begin equilibration. After completing equilibration, the assay will automatically begin acquiring baseline measurements (as outlined in your instrument protocol).
Once the final measurement command in the instrument protocol is completed, the Remove Plate and Cartridge dialog will appear. Click Continue to eject the sensor cartridge and cell plate. Set aside for later analysis if necessary (example - cell count normalization).
Save or transfer the assay result file to a shared network drive or USB drive and open using Wave Desktop software on your PC for data analysis.

+ Related Support Material

+ Analyze XF Assay Results

This section focuses on viewing & analyzing assay result data using Wave Desktop and Report Generator software.

Wave Desktop is the data analysis software for result files generated from any XF, XFe and XFp Analyzer. Transform complex cellular metabolism data into publishable results using Wave Desktop's flexible analysis views, embedded reporting tools, and other powerful analytical capabilities.

XF Report Generators are Microsoft Excel macro files that automatically calculate the parameters of each Seahorse XF Assay Kit and present result data in a one-page, customizable Summary Report.

Select a topic below to learn more:

PC Specs &
Compatibility

Types of
Data

Data Display
Options

Analysis
Views

XF Report
Generators

PC Specs & Compatibility

Wave Desktop is the assay design & data analysis software for all Seahorse Analyzers and supports:

Analysis of data files from all Seahorse Analyzers (XFe96, XFe24, XFp, XF96 and XF24).
Create and customize assay templates for XFe96, XFe24 & XFp Analyzers.
Export data to (1) Microsoft Excel, (2) GraphPad Prism, or (3) XF Report Generators.
Supports Microsoft Excel (32 & 64-bit) for both Windows and Macintosh PCs.

It is always encouraged to update to the latest version of Wave Desktop software. The latest release of Wave Desktop software is version 2.6 (March 2018). Wave Desktop software can be installed on any PC with Windows 7 Operating Systems or later.

Below you will find the PC specs and compatibility detail for Wave Desktop 2.6 software:

Computer	Specifications
Windows PC	Operating System: Windows 7, 8.1, and 10
	Processor: Intel Core i3 (or better)
	Hard Disk Space: 175 GB
	System Memory (RAM): 4 GB (minimum*)
	Screen Resolution: 1280 x 800 (minimum)
	Supported Excel Versions: 2010, 2013 & 2016
Macintosh PC (requires use of a virtual machine)	Operating System: Mac OSx 10.11 or higher
	Virtual Machine: Parallels 12 & Windows 7, 8.1 and 10
	Processor: Intel Core i3 (or better)
	Hard Disk Space: 175 GB
	System Memory (RAM): 4 GB (minimum*)
	Screen Resolution: 1280 x 800 (minimum)
	Supported Excel Versions: 2011 & 2016

* For the optimal software experience, 8 GB (or higher) System Memory (RAM) is recommended.

+ Related Support Material

Types of Data

Cellular oxygen consumption (respiration) and proton excretion (glycolysis) cause rapid, easily measurable changes to the concentrations of dissolved oxygen and free protons. Agilent Seahorse XF Analyzers measure concentrations of dissolved oxygen and free protons in real-time by isolating an extremely small volume (about 2 μL) of medium above a monolayer of cells within a microplate then calculate the OCR and ECAR, respectively.

Using Wave Desktop software, you can easily access & review these data:

Rate Data is the primary output of the XF analyzer.

Oxygen Consumption Rate (OCR): A quantitative measurement of oxygen consumption in the well, an indicator of mitochondrial respiration, reported in picomole/minute (pmol/min) vs. time.
Oxygen Consumption Rate (OCR) data displayed in Rate mode (right).
Extracellular Acidification Rate (ECAR): A qualitative measurement of proton extrusion in the extracellular medium, reported as milli-pH/minute (mpH/min) vs. time.
Extracellular Acidification Rate (ECAR) data displayed in Rate mode (right)
Proton Efflux Rate (PER): A quantitative measure of extracellular acidification that accounts for media buffering capacity and plate geometry. Reported in picomole/minute (pmol/min) vs. time. Only available in post-run assay results, not during assay runtime.

See chapter 3 in the Wave User Guide for more information about the types of data available in Wave software.

Level Data is used to calculate rate data and can also be used for diagnostic purposes.

Oxygen Concentration (mmHg): As cells (or other biological material) consumes oxygen during a measurement, oxygen tension will decrease. This decrease in oxygen tension is used to calculate the rate of oxygen consumption (OCR). You can view oxygen tension level data on the Overview analysis view using the Y1 drop-down menu.
Oxygen tension (O₂) data displayed as mmHg vs. time in Level mode (right)
Proton Concentration (mpH): As cells (or other biological material) generate protons during a measurement, proton concentration will increase. The concentration of free protons in the microchamber is measured throughout an XF assay and calculated as the rate of extracellular acidification (ECAR). You can view mpH level data on the Overview analysis view using the Y1 drop-down menu.
Proton Concentration (mpH) data displayed in Level mode (right)

+ Related Support Material

Data Display Options

Wave provides a standard set of graphing options to view & interpret assay result data. Depending on the type of analysis view selected, Wave automatically calculates and graphs result data as one of the following:

Kinetic Graph

A Kinetic Graph is the most common way to display rate data from XF Analyzers. A kinetic graph displays the rate on the y-axis, and time on the x-axis. During an assay, data is acquired and plotted in real-time as a kinetic graph. Kinetic graphs can be found in the Quick View and Overview analysis views in Wave software.

Energy Map

Another common way to graph XF result data is an Energy Map (scatter plot), where Oxygen Consumption Rate (OCR) is always plotted on the y-axis, and acidification data (ECAR or PER) is always plotted on the x-axis. Use the Measurement drop-down menu to select a rate measurement to display on the energy map for each group. Use the Rate drop-down menu to change the x-axis rate to PER. The energy map graph option can be found in the Quick View and OCR vs. ECAR analysis views.

Plate Map

The Plate Map displays rate data for the selected rate measurement of each assay well. Plate Maps are always displayed in the upper right corner of Wave while running an assay, and on the Quick View, Overview, and OCR vs. ECAR analysis views. The Quick View has a button to display the Plate Map, which is hidden by default.

The Plate Map in the Quick View and OCR vs. ECAR analysis views displays two rates: Oxygen Consumption Rate (OCR - top) and acidification data (either ECAR or PER - bottom). The Plate Map on the Overview analysis view displays 1 rate - OCR, ECAR, or PER.

When opening a new analysis view, the Plate Map shows data for rate measurement 1 by default. Use the Rate drop-down menu to display data for another rate measurement during the assay.

Bar Graph

The Bar Graph is available on the Overview analysis view only (below the Plate Map), and displays the average rate for each group for the selected measurement. Use the Display drop-down menu to change the rate display from Group (average) to Well (individual well) mode.

The Bar Graph can be used for identifying outliers, optimal FCCP concentration, optimal cell seeding density, or other trends that may not be apparent when measurement data is plotted as a kinetic graph or scatter plot.

Group List

The Group List is the legend for the data plotted in the kinetic graph or scatter plot.

Use the Group List to:

Hide or show groups from the graphed data by double-clicking the group name.
Check the Details box in the upper-right corner of the Group List to display group statistics. Statistics are displayed as average and error for the selected rate measurement. Select a different rate measurement to display group statistics for that rate.
Assay wells that have been turned OFF on the Plate Map are not included in the calculated group statistics.
When a group is hidden, the mean and standard deviation of the group will be: Mean: 0.00; Standard Deviation: 0:00.

See chapter 3 in the Wave User Guide for more information about the types of data available in Wave software.

+ Related Support Material

Analysis Views

There are 4 customizable analysis views that can be added to an assay result file in Wave Desktop. Each analysis view can be added to an assay result file multiple times using the Add View button in the top-level ribbon menu.

Quick View

Quick View is the default analysis view displayed when opening a new assay result file. Quick View simultaneously displays a kinetic graph of OCR vs time, ECAR vs time, and an energy map of OCR vs. ECAR.

Overview

Overview displays a kinetic graph of rate (OCR, ECAR, PER, or PPR) versus time. The selected rate is displayed on the y-axis, and time is displayed on the x-axis. Group statistics (average rate and error) for each measurement can be displayed by checking the Details box in the Group List below the kinetic graph. Overview is the most versatile analysis view in Wave software for routine analysis functions. To display Overview, click Add View and select Overview from the list of views.

Tip: Add multiple Overview analysis views by repeating the process of Add View > Overview. This gives greater flexibility for tailoring your result data presentation to show specific groups, responses, or comparisons between groups.

OCR vs. ECAR

The OCR vs. ECAR view displays an energy map with the OCR on the y-axis and (by default) ECAR on the x-axis. Use the Rate drop-down menu to change the rate displayed on the x-axis to either PER or PPR. OCR is always displayed on the y-axis, and cannot be changed. To display OCR vs. ECAR, click Add View and select OCR vs. ECAR from the list of views.

Data

The Data view contains all data associated with the assay result file organized into 7 tabs:

Group Data: Average rate data (OCR, ECAR, PER, or PPR) and error for each group, ordered by measurement number.
Rate: Individual well rate data (OCR, ECAR, PER or PPR) ordered by measurement number. Error values are not displayed.
Level Data: Individual well level data (O₂ and pH) ordered by measurement number.
Raw: Raw measurement data, including O₂ and pH light emission values, reference values, well and environment temperature recorded each measurement. This tab is most-commonly used by Tech Support, not for routine data analysis.
Calibration: O₂ and pH calibration results organized by well.
Calibration View: O₂ and pH calibration results for each assay well displayed as a plate map.
Event Log: Assay information such as analyzer serial number, software version, plate & barcode lot numbers, and other settings during the assay. Also provided is a sortable table of protocol commands used during the XF assay, including command name, timing, and outcome.

To display the Data view, click Add View and select Data from the list of views. The Group Data tab is displayed by default when opening the Data view.

See Chapter 3 in the Wave User Guide for more detailed information about each analysis view, including recalculating data as a % of baseline, normalize rate data to a biological parameter (i.e. cell number), flag assay wells on the plate map, and other key analysis functions & features.

+ Related Support Material

XF Report Generators

Agilent Seahorse XF Report Generators are Microsoft Excel macro files that automatically calculate the parameters of each Seahorse XF Assay Kit and present result data in a one-page, customizable Summary Report.

Wave provides a one-click direct export of result data to the XF Report Generators, any modifications to your result data in Wave, such as excluded assay wells or normalized rate data, will be incorporated in the exported & analyzed Report Generator Excel file. Click the Export button in the top-level ribbon menu to display the list of available XF Report Generator export options.

Why use XF Report Generators?

More efficient & consistent data analysis - transform raw kinetic data into interpretable results and eliminate repetitive manual calculations and data reduction.
Summarize XF result data in seconds - data is presented as an organized, customizable easy to understand report.
Scalable to meet your analysis needs - import and analyze up to 5 replicate result files at once using the Multi-File XF Report Generators.
Simpler collaboration - review and reanalyze result data in the Report Generators without any special software programs or licenses.
Supports analysis of:

Data files generated on Seahorse XFe, XF and XFp Analyzers.
Microsoft Excel 2010, 2013, and 2016 on Windows PCs.
Excel for Mac 2011 and 2016 for Apple computers.

XF Report Generators - Download pages	Single-File Analysis	Multi-File Analysis
Seahorse XF Cell Energy Phenotype Test	Single-File User Guide	Multi-File User Guide
Seahorse XF Cell Mito Stress Test	Single-File User Guide	Multi-File User Guide
Seahorse XF Real-Time ATP Rate Assay	Single-File User Guide	Not Available
Seahorse XF Glycolytic Rate Assay	Single-File User Guide	Multi-File User Guide
Seahorse XF Mito Fuel Flex Test	Single-File User Guide	Not Available
Seahorse XF Glycolysis Stress Test	Single-File User Guide	Not Available

+ Related Support Material

+ Beyond the Basics

+ Index

Need more information?

Worldwide Technical Support

Email: cellanalysis.support@agilent.com

USA and Canada
1-800-227-9770; select option 3 then 8

Europe
UK: 0800 096 7632
Germany: 0800 180 66 78
Netherlands: 0800 022 7243
Other EU: +45 3136 9878

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