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

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

Note: All XFp assay kits and consumables are compatible with the XF HS Mini Analyzer.

1.1 Required Materials from Agilent

1.2 Other Required Materials Provided by the User

1.1 Required Materials from Agilent

1.2 Other Required Materials Provided by the User

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.

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 miniplates, XF HS miniplates, or XF HS PDL miniplates and hydrating an 8-well sensor cartridge.

Note: See workflow diagram for an overview of the steps involved in running an XF HS Mini assay.

Select a workflow step to display help content.

2.1 Choose Cell Seeding Densities

2.2 Hydrate Cartridge

2.2 Hydrate Cartridge

2.3 Design Experiment

2.3 Seeding Cells

2.4 Seeding Cells

2.4 Seeding Cells

2.4.1 XFp miniplates

2.4.2 XF HS miniplates

2.5 Prepare XFp PDL or XF HS PDL miniplates (suspension cells only)

2.1 Choose Cell Seeding Densities

2.2 Hydrate Cartridge

Basic procedures for hydrating the 8-well sensor cartridge

Note: XFp sensor cartridges are compatible with the XF HS Mini analyzer

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.
• The sensor cartridge hydration protocol continues in section 3.1 of the Learning Center, please continue reading

• 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.

2.4 Seeding Cells

2.3 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 Cell Culture Microplate Cell Culture Miniplatep, XF HS, XFp PDL, or XF HS PDL miniplates in conjunction with an Sensor Cartridge. XFe96 Sensor Cartridge. 8-well 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, XFp8-well sensor 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:

2.4.1 XFp miniplates

Plate type	Final adherent cell seeding volume	Final seeding density (Example only)	Converted to mL
XFp miniplates / XFp PDL miniplates	80 μL	1.0 × 104 cells / 80 µL	1.25 × 105 cells/mL

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 80 µL of cell suspension per well. Do not add cells to background wells A and H. If bubbles are observed at the bottom of the well, the plates may be centrifuged 1 to 2 minutes at 200 × g to remove them.
• 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

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.

Select a workflow step to display help content.

3.1 Prepare Cartridge and Assay Medium

3.2 Wash Cells

3.3 Assemble Solutions

3.4 Load Injection Solutions

3.2 Seeding Suspension Cells

3.2.1 XFp PDL miniplates

3.2.2 XF HS PDL miniplates

3.3 Washing Adherent Cells

3.3.1 XFp PDL miniplates

3.3.2 XF HS PDL miniplates

3.4 Assemble Solutions

3.5 Load Injection Solutions

3.1 Prepare Cartridge and Assay Medium

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 assay Cell 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 kit Cell 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	Seahorse XF DMEM mediuma,b / 103575-100 or Seahorse XF RPMI mediuma,b / 103576-100	-	97.0 mL	9.70 mL
Seahorse XF Glucose (1.0 M solution) / 103577-100	10 mM	10 μL	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.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 Preparation using alternative types of XF media Preparation using alternative types of XF media Preparation using alternative types of XF media Preparation 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

3.2 Seeding Suspension Cells

Basic procedures for seeding suspension cells on XF HS PDL miniplates and XFp PDL miniplates

Note: For additional information on seeding suspension cells in XF HS PDL miniplates, please follow the instructions found in the Related Support Material below.

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 Culture Microplate, transfer an appropriate volume of cell suspension from the growth vessel to a conical tube.
• For one XFp Miniplate, XF HS Miniplate or XF HS PDL Miniplate, 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.

Overview of the steps required for seeding suspension cells on XFp PDL or XF HS PDL miniplates.

3.4 Load Injection Solutions

3.5 Load Injection Solutions

• 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.
• 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	20 µl	55 µl	15 µM	1.5 µM
Port B Rotenone + Antimycin A	22 µl	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

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.

• 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.

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

This section focuses on performing initial cell characterization using the 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 XF HS Mini Analyzer. For more detailed information about performing an assay, please consult the XF HS Mini User Guide.

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.

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.

In the Analyze XF Assay Results section, you will learn the basics of Agilent Seahorse Analytics and how to use Seahorse Analytics to analyze your XF Real-Time ATP Rate assay result file. If you are looking for help content for Wave Desktop and Report Generators, please click here.

Seahorse Analytics is a web-based software application that provides desktop-like interactivity with an easy-to-learn interface, allowing you to analyze result data from your Agilent Seahorse XFe, XFp, and XF HS Mini Analyzer from any computer, from anywhere in the world. As long as you have internet access, you can analyze your Seahorse data with Seahorse Analytics.

Please Note: For XF96 users you must first convert your .xfd result file to the assay result file format using Wave Desktop software, .xfd result files cannot be analyzed in Seahorse Analytics.

Select a topic below to learn more:

5.1 Introduction

5.2 Analysis Views

5.3 Types of Data

5.4 Data Display Options

5.5 Export & Share Data

5.1 Introduction

Agilent Seahorse Analytics is the go-to data analysis software for your Seahorse Analyzers Analyzer, enabling you to easily import, analyze, report, and share your results with your team or collaborators.

Basic information about Agilent Seahorse Analytics:

Specification	Detail
Used For	Data analysis
Seahorse Data File Compatibilty	XFe96 assay result file
	XFe24 assay result file
	XFp assay result file
	XF HS Mini assay result file
Internet Browser Compatibility	Google Chrome
	Safari
	Mozilla Firefox
	Microsoft Edge Use of Internet Explorer is strongly discouraged
Export Options	Microsoft Excel (.xlsx)
	Graphpad Prism (.pzfx)
	Image File (.JPG and .PNG)

Getting familiar with the Seahorse Analytics User Interface

Home View:

After you register and/or log-in to your Seahorse Analytics account, you will find yourself on the Home view (pictured below). To get back to the Home view, click the Home button in the upper-left corner of the dark blue navigation ribbon.

1. The data files list on the Home view displays the most recent five (5) files you have imported, edited, and/or accepted from a file share.
2. Below the files list, you will see My Analyzers, where you can assign one or more Seahorse XF Analyzers to your profile.
3. To import a data file from the Home view, click the small File Upload button in the upper-right corner above the files list.

Files View:

Click the Files button in the upper-left corner of the dark blue ribbon at the top of the application to display all data files you have imported to your Seahorse Analytics account.

1. On the left of the Files view, you will see any custom folders you have created for your account.
2. The list of files and accompanying file meta data (i.e. last modified date, instrument type, etc.) is seen in the middle of the Files view.
3. In the upper-right corner of the Files view, you will see the File Upload button, allowing you to import data files to your account. You will find the Create Folder button, which will create a new custom folder you can assign data files to. You will also find a search field that allows you to perform keyword searches of the data files in your account. The search function looks for keyword matches in the file name, categories, last modified date, and instrument type.

Resource View:

Click Resources in the top-level dark blue ribbon to access a collection of frequently used resources to support your Agilent Seahorse XF assay workflow and contact information for the Agilent Cell Analysis support team.

Settings and User Information:

Other file management features:

To the right of each file in your account, you will notice a small 3-dot icon (pictured below).

5.2 Analysis Views

A Seahorse Analytics analysis view is a collection of widgets (graphs) in a single page. There are 3 different analysis view categories – standard analysis views, custom analysis views, and assay kit companion analysis views.

• The standard analysis view contains basic widget options such as kinetic graph, scatter plot and bar chart, which are used to view OCR, ECAR, PER for a specific rate measurement or series of rate measurements (i.e. kinetic graph).
• The custom analysis view list displays all custom analysis views containing user-selected defined widgets. Any type of widget can be added to a custom analysis view.
• The assay kit companion analysis view list shows analysis views where the widgets on each view represent defined parameters of the selected Agilent Seahorse XF assay kit. The assay kit companion analysis views can be used for data analysis of files generated using respective supported Agilent Seahorse XF assay workflows and protocols.

Expand the Views list to display the list of analysis views applied to an assay result file or to add a new analysis view (pictured below).

Other important points:

• Not all XF assay workflows can be analyzed using Seahorse Analytics. You can export your result data to Microsoft Excel or GraphPad Prism for custom analysis needs.
• Each widget has its own plate map that controls the graphed data for that widget. To edit the graphed data, open the widget editor view by double-clicking the widget. The graphed data is controlled using the functions seen in the ribbon above the graph and using the plate map to the right of the graph. To make file-level changes go to the Modify view.
• There are times when you make wish to make small modifications to your assay result file. You can access the modify functions from any analysis view by clicking the Modify button found in the upper-right corner in the ribbon. Changes to a file using Modify will affect all widgets/analysis views in your result file (i.e. removing an outlier well, changing group color, or renaming a group).

Blank and Custom Analysis Views: The custom analysis view feature provides ultimate data analysis flexibility. You can save any analysis view as a custom view, however this example shows you how to create a custom analysis view starting from a Blank view. The series of steps below also highlights how to automate routine steps in your analysis workflow using the blank/custom view feature, improving workflow consistency and saving time.

Assay Kit Companion Analysis View Information:

1. Assay Kit Companion Analysis View » XF Cell Mito Stress Test: To calculate and display the XF Cell Mito Stress Test assay parameters in a single analysis view:

1. On the Add View window, click the Assay Kit Companion View list to expand the list of options.
2. Click the XF Cell Mito Stress Test analysis view to display assay parameter widgets.
3. Important: For XF Cell Mito Stress Test assays with 4 injections, you must identify the oligomycin injection using the drop-down menu seen above the widgets before you can add this analysis view. This oligomycin injection selection plays a critical role in correctly calculating assay parameters; incorrect oligomycin injection selection will result in incorrect widget calculations and graphs.
4. Select the desired parameter widgets (e.g. basal respiration, maximal respiration, etc.) to add to your analysis view. A blue outline around the widget indicates it is selected. If the Acute Response widget is greyed-out, then it cannot be selected as this parameter can only be calculated for XF Cell Mito Stress Test assays with an acute injection.
5. Turn OFF/ON groups in the group list if necessary, then click Add View

You can also add individual XF Cell Mito Stress Test assay parameter widgets (i.e. Spare Respiratory Capacity) to an existing analysis view:

Assay Parameter	Equation
Non-Mitochondrial Oxygen Consumption	Minimum rate measurement after Rotenone/antimycin A injection
Basal Respiration	(Last rate measurement before first injection) – (Non-Mitochondrial Oxygen Consumption)
Maximal Respiration	(Maximum rate measurement after FCCP injection) – (Non-Mitochondrial Oxygen Consumption)
Proton Leak	(Minimum rate measurement after Oligomycin injection) – (Non-Mitochondrial Oxygen Consumption)
ATP-Production Coupled Respiration	(Last rate measurement before Oligomycin injection) – (Minimum rate measurement after Oligomycin injection)
Spare Respiratory Capacity	(Maximal Respiration) – (Basal Respiration)
Spare Respiratory Capacity as a %	(Maximal Respiration) / (Basal Respiration) × 100%
Acute Response	(Last rate measurement before Oligomycin injection) – (Last rate measurement before acute injection)
Coupling Efficiency	(ATP-Production Coupled Respiration) / (Basal Respiration) × 100%

4.2. Assay Kit Companion Analysis View » XF Real-Time ATP Rate Assay: Follow the steps below to calculate and display the XF Real-Time ATP Rate Assay parameters in a single analysis view. Please note, buffer factor must be properly configured to add this analysis view and widgets from this analysis view to your assay result file.

1. On the Add View window, click the Assay Kit Companion View list to expand the list of options.
2. Click the XF ATP Rate Assay analysis view to display assay parameter widgets.
3. For Induced XF ATP Rate assays (1 or 2 injections prior to standard injections of oligomycin and rotenone/antimycin A), you must identify whether oligomycin injection was injection 2 or injection 3 using the drop-down menu seen above the widgets to add this analysis view. This oligomycin injection selection plays a critical role in correctly calculating assay parameters; incorrect oligomycin injection selection will result in incorrect widget calculations and graphs. For more information on these two workflows please consult the XF Real-Time ATP Rate Assay User Manual.
4. Select the desired parameter widgets to add to your analysis view. A blue outline around the widget indicates it is selected. If widget(s) are greyed out, this means they cannot be selected as the assay file does not have the minimum number of injections required to calculate the parameter(s).
5. Turn OFF/ON groups in the group list if necessary, then click Add View

You can also add individual XF ATP Rate Assay parameter widgets (i.e. ATP Production Rate Basal) to an existing analysis view:

Standard XF Real-Time ATP Rate Assay

Assay Parameter	Equation
mitoATP Production Rate (Basal)	[(Last OCR rate measurement before first injection - Minimum OCR rate measurement after oligomycin but before Rot/AA injection) x 2 x (P/O)]
glycoATP Production Rate (Basal)	Last glycoPER measurement before first injection
Total ATP Production Rate (Basal)	(mitoATP Production Rate) + (glycoATP Production Rate)
XF ATP Rate Index (Basal)	(mitoATP Production Rate) / (glycoATP Production Rate)

Induced XF Real-Time ATP Rate Assay:

Assay Parameter	Equation
mitoATP Production Rate (Basal)	[(Last OCR rate measurement before first injection - Minimum OCR rate measurement after oligomycin but before Rot/AA injection) x 2 x (P/O)]
glycoATP Production Rate (Basal)	Last glycoPER measurement before first injection
Total ATP Production Rate (Basal)	(mitoATP Production Rate) + (glycoATP Production Rate)
XF ATP Rate Index (Basal)	(mitoATP Production Rate) / (glycoATP Production Rate)
mitoATP Production Rate (Induced)	(Average OCR rate measurement after acute injection and before oligomycin injection) - (Minimum OCR rate measurement after oligo & before Rot/AA injection)] x 2 x (P/O)
glycoATP Production Rate (Induced)	Average of the glycoATP Production Rate measurements after the acute injection and before next injection.
Total ATP Production Rate (Induced)	mitoATP Production Rate (Induced) + glycoATP Production Rate (Induced)
XF ATP Rate Index (Induced)	mitoATP Production Rate (Induced) / glycoATP Production Rate (Induced)

5.3. Assay Kit Companion Analysis View » XF T Cell Activation Assay: To calculate and display the XF T Cell Activation assay parameters in a single analysis view:

1. On the Add View window, click the Assay Kit Companion View list to expand the list of options.
2. Click the XF T Cell Activation Assay analysis view to display assay parameter widgets.
3. Check the Baseline checkbox to calculate PER data displayed in each widget as a percent (%) of the baseline rate measurement, which is the last rate measurement before the activator injection. For quantitative data, leave the Baseline checkbox unchecked to calculate PER in units of (pmol/min).
4. Ensure the correct injection position is selected in the Activator Injection drop-down menu.
5. Select the desired parameter widgets to add to your analysis view. A blue outline around the widget indicates it is selected.
6. Turn OFF/ON groups in the group list if necessary, then click Add View

You can also add individual XF T Cell Activation Assay parameter widgets (i.e. Maximum Rate) to an existing analysis view:

The table below describes the XF T Cell Activation Assay parameter calculations:

Assay Parameter	Equation
T Cell Activation	PER (proton efflux rate) data displayed as a kinetic graph
Maximum Rate	Calculated in Seahorse Analytics using a nonlinear least-squares curve fitting algorithm using rate measurement data after the activator injection and before the 2-DG injection. For more information regarding this calculation method, please refer to: H. J. Motulsky, "Exponential plateau", GraphPad Curve Fitting Guide. Accessed 20 May 2020. https://www.graphpad.com/guides/prism/8/curve-fitting/REG_Exponential-plateau.htm
Area Under the Curve	Calculated in Seahorse Analytics using rate measurement data after the activator injection and before the 2-DG injection. AUC values only include peak values above baseline. For more information regarding this calculation method, please refer to: H. J. Motulsky, "How to: Area under the curve", GraphPad Statistics Guide. Accessed 20 May 2020. https://www.graphpad.com/guides/prism/8/statistics/stat_area_under_the_curve.htm

6.4. Assay Kit Companion Analysis View » XF Glycolytic Rate Assay: Follow the steps below to calculate and display the XF Glycolytic Rate Assay parameters in a single analysis view. Please note, buffer factor must be properly configured to add this analysis view and widgets from this analysis view to your assay result file.

1. On the Add View window, click the Assay Kit Companion View list to expand the list of options.
2. Click the XF Glycolytic Rate Assay analysis view to display assay parameter widgets.
3. For Induced XF Glycolytic Rate Assay (1 or 2 injections prior to standard injections of rontenone/antimycin A and 2-DG), you must identify the Rotenone/Antimycin-A injection using the drop-down menu seen above the widgets before you can add this analysis view. This Rotenone/Antimycin-A injection selection plays a critical role in correctly calculating assay parameters; incorrect Rotenone/Antimycin-A injection selection will result in incorrect widget calculations and graphs. For more information about these 2 workflows, please consult the XF Glycolytic Rate Assay User Manual.
4. Select the desired parameter widgets to add to your analysis view. A blue outline around the widget indicates it is selected. If widget(s) are greyed out, this means they cannot be selected as the assay file does not have the minimum number of injections required to calculate the parameter(s).
5. Turn OFF/ON groups in the group list if necessary, then click Add View

You can also add individual XF Glycolytic Rate Assay parameter widgets (i.e. Basal Glycolysis) to an existing analysis view:

Standard XF Glycolytic Rate Assay

Assay Parameter	Equation
Basal Glycolysis	Last glycoPER measurement before first injection
Basal Proton Efflux Rate (PER)*	Last PER measurement before first injection
% PER from Glycolysis (Basal)	(Basal Glycolysis) / (Basal PER) x 100%
* Denotes this parameter is not reported individually in Seahorse Analytics but used to calculate other reported parameter values.

Induced XF Glycolytic Rate Assay

Assay Parameter	Equation
Basal Glycolysis	Last glycoPER measurement before first injection
Basal Proton Efflux Rate (PER)*	Last PER measurement before first injection
% PER from Glycolysis (Basal)	(Basal Glycolysis) / (Basal PER) x 100%
Compensatory Glycolysis	Maximum glycoPER measurement after Rot/AA injection
Induced Glycolysis	Average of the glycoPER measurements after the induced assay injection and before Rot/AA injection
Induced PER*	Average of the PER measurements after the induced assay injection and before next injection
% PER from Glycolysis (Induced)	(Induced Glycolysis) / (Induced PER) x 100%
* Denotes this parameter is not reported individually in Seahorse Analytics but used to calculate other reported parameter values.

Editing graphed widget data: To customize the data graphed for a widget, double-click the widget to open the widget-editor view. There are different ways you can customize the data graphed for a specific widget. It is important to remember the plate maps for each widget are independent, meaning the changes you make on the widget-editor view will ONLY be applied to that respective widget.

The functions in the ribbon above each graph provides editing functions specific for that widget type, such as changing the rate data type, toggling normalization on/off, or looking at data well-by-well rather than as group averages. Refer to the different widget descriptions for more information on these functions and how to use them.

The plate map to the right of the graph allows you to include or exclude specific assay wells from calculations and graphed data for the selected widget type.

The group list below the widget shows you group average and error data for the selected widget type. You can double-click a group name to show or hide all data in that group from the widget graph. Assay wells turned off on the plate map are not included in the values seen in the group list. When a group is excluded from the graphed data, the group will appear grayed-out as seen in the “Control” group in the image below.

Define Buffer Factor

A common analysis workflow is to define buffer factor for your assay media and background wells in an assay result file. This information is required to calculate Proton Efflux Rate (PER), which is calculated and displayed in many widgets on several assay kit companion analysis views in Seahorse Analytics (i.e. XF T Cell Activation assay). Attempting to add an analysis view to an assay result file that does not have buffer factor properly configured will result in an error message (pictured below). Once buffer factor is properly assigned to your the media and background wells in the assay result file, the desired widgets and/or analysis views can be opened.

Steps to resolve the buffer factor error – If you see the error pictured above when adding the first analysis view to a data file, start at step 1 below. If you already have an analysis view open in the data file, start from step 3.

1. Click OK to dismiss the error dialog.
2. On the Add View window, select Standard Views > Quick View and choose the OCR kinetic graph widget.

5.3 Types of Data

Cellular oxygen consumption (respiration) and proton excretion (glycolysis) cause rapid, easily measurable changes to the concentrations of extracellular oxygen and protons. Agilent Seahorse XF Analyzers measure changes in concentration of extracellular oxygen and protons in real-time. Using Seahorse Analytics, you can easily access & review the following types of data:

1. Rate Data is the primary output of all Seahorse XF analyzers. The 3 types of rate data calculated & reported in Seahorse Analytics are:

2. Level Data is used to calculate rate data and used for data quality assessment – typically the first step of the XF analysis workflow.

5.4 Data Display Options

Agilent Seahorse Analytics provides a selection of graph types to analyze & interpret assay result data depending on the type of analysis view preferred. For more information about creating and customizing analysis views, please see the Analysis Views section.

1. Widget Types – Kinetic Graph: A kinetic graph is the most common way to display XF result data, where your x-axis is time (in minutes) and your y-axis is the rate of change in concentration of the analyte measured (O₂ or H+). The two types of data you can display on the y-axis of the kinetic graph widget are (1) Rate data – as oxygen consumption (OCR), extracellular acidification (ECAR), proton efflux (PER) and (2) Level Data – as oxygen tension (mmHg), or proton concentration [mpH].

• Add Widget > Standard Graphs » Kinetic Graph:
  Use the Standard Graphs » Bar Chart widget to create a bar chart of a selected rate type (OCR, ECAR, PER) and measurement.

2. Widget Types – Bar Chart: A bar chart is another common way to present XF data, aiding users in routine XF analysis such as determining optimal FCCP concentration and cell seeding density. Seahorse Analytics provides a wide variety of bar chart options to add to an analysis view, and many are assay specific. For more information about specific bar chart widgets please refer to the specific assay kit companion analysis view here or in Seahorse Analytics.

• Add Widget > Standard Graphs » Bar Chart:
  Use the Standard Graphs » Bar Chart widget to create a bar chart of a selected rate type (OCR, ECAR, PER) and measurement.

3. Widget Types – Energy Map: An Energy Map widget is an X & Y scatter plot, allowing comparison of two types of rate data for a selected rate measurement for all or selected groups. In addition to the Energy Map widget described below, there are several additional scatter plot widget options you can add to your analysis views that are assay type dependent and defined in the Analysis Views section.

• Add Widget > XF ATP Rate Assay » XF ATP Rate Index: The XF ATP Rate Index is currently found in the XF ATP Rate Assay widget list. The XF ATP Rate Index widget plots the ratio of basal mitoATP production rate to basal glycoATP production rate as an empty circle marker, and plots the ratio of the average of the induced mitoATP production rates to the average of the induced glycoATP production rates for each group as a filled circle marker (only when applied to an induced assay result file – widget pictured below shows data from an induced XF ATP rate assay). Please also note that buffer factor must be properly assigned to the assay media, background wells, and assay groups before you can calculate this widget data.

5.5 Export & Share Data

Export your data to a Microsoft Excel or GraphPad Prism file or share your result data and insight with a collaborator directly using Seahorse Analytics.

Data Export:

There are several ways you can export data from Seahorse Analytics to a Microsoft Excel, GraphPad Prism, or image file.

Export all rate data

You can create an Excel and Prism file containing all rate data for the selected file. This export will not include any parameter calculations (i.e. Spare Respiratory Capacity) that you have calculated in an analysis view, you will need to export that data individually from each widget.

How is this export feature used? This type of data export allows users to easily transfer all rate data (for OCR, ECAR, and PER) data into Prism or Excel to create high-quality or customized figures, perform statistical analysis, and other analytical functions not offered in Seahorse Analytics.

Export select data from individual widgets: You can export individual widget data to an Excel and Prism file containing data for the selected widget. The data in the export file will match exactly how you formatted the widget. For example, if you configure a widget to display basal respiration in well mode, the Prism export file will contain individual well values for basal respiration data for each group. If you configure a widget to display basal respiration in group mode, the Prism export file will show the average group value and error value, not individual well values.

How to export all rate data:

1. From any analysis view or widget editor view, look for the small cloud button in the upper-right corner of the widget.
2. Move your mouse cursor over the cloud button to display the Image and Data export options.
3. Slide your cursor over Image to export your widget as a PNG or JPG file, or Data to export your widget as an Excel or Prism file.
4. Data in the export file will exactly match how you formatted your widget. In this example, the Prism export file contains basal respiration values per well for both groups on the plate.

How is this export feature used? This type of data export allows users to easily export select rate or parameter data as an image file, or as a Prism or Excel file. The Prism and Excel export allow you to get exactly the data you want to further analyze (i.e. create high-quality or customized figures, perform statistical analysis, and other analytical functions that are not offered in Seahorse Analytics) rather than exporting all data and trying to find the specific information you need.

How is this sharing feature used? The share feature allows you to send an assay result file to someone with, or without, a Seahorse Analytics account directly through the application. The assay result file you choose to share will create a copy of that data file in the recipient’s account, and the widget layout, selection and analysis views will appear exactly how you formatted them prior to the file share. The recipient can make modifications to the shared file, but your copy of the file will remain unchanged.

This section focuses on viewing & analyzing assay result data using Wave Desktop and Report Generator software. To display help content for Seahorse Analytics, click here.

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:

5.1 PC Specs & Compatibility

5.2 Types of Data

5.3 Data Display Options

5.4 Analysis Views

5.5 XF Report Generators

5.1 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 2.6.1 (April 2019). 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.

5.2 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.

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

5.3 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.

5.4 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.

5.5 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

This section of the XF Learning Center will provide an introduction to various topics on using XF Analyzers, including a range XF assay kits and applications, alternative assay conditions and sample types, as well as normalization and analysis of XF data.

Select a topic below to learn more:

6.1 XF Kits & Applications

6.1 XF Kits & Applications

6.2 Normalization of XF Data

6.2 Fatty Acid Oxidation Assays

6.3 Permeabilized Cells & Iso Mitos

6.3 Permeabilized Cells & Iso Mitos

6.4 Hypoxia and Spheroids

6.4 Hypoxia and Islets

6.1 XF Assays and Applications

Agilent XF Assay Kits and reagents are developed specifically for use with each XF Analyzer to ensure reliability and consistency of results. Seahorse XF kits and reagents help simplify running an XF assay by providing pre-calibrated, pre-tested reagents for measuring valuable functional metabolic parameters including cellular ATP production rates, mitochondrial function, glycolytic activity and substrate oxidation in living cells, permeabilized cells and isolated mitochondria.

Note: All XFp assay kits and consumables are compatible with the XF HS Mini Analyzer.

The materials below provide information and methods for performing a wide range XF Assays.