Agilent Seahorse XFp T Cell Metabolic Profiling Kit

User Guide
Kit 103771-100

For use with Agilent Seahorse XF HS Mini or XFp Extracellular Flux analyzers and with T cells or
NK cells.

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Manual Part Number
5994-3494EN Rev. C

EditionSecond edition, March 2024
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Contents
1

Introduction
Assay Background 5
XF T Cell Persistence assay 5
XF T Cell Fitness assay 6
XF NK Cell Metabolic Profiling assay
Glossary

8

References
2

8

Kit Information
Kit Contents

3

6

9

Kit Shipping and Storage

9

Additional Required Items

10

Assay Workflow for XF HS Mini and XFp Analyzer
One Day Prior to Assay (Day 1) 12
Prepare the sensor cartridge and HS or XFp PDL miniplate 12
Design and upload the assay template to the XF HS Mini or XFp analyzer

13

Day of Assay (Day 2) 14
Prepare the cartridge 14
Prepare the assay medium 14
Prepare and seed cells in the Seahorse HS or XFp PDL miniplates 15
Prepare the compound solutions and load the injection ports 17
Running the assay on the XF HS Mini analyzer or XFp analyzer 18
Data Analysis
419Frequently Asked Questions

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Agilent Seahorse XFp T Cell Metabolic Profiling Kit User Guide

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Introduction
Assay Background 5
Glossary 8
References 8

Assay Background
The Agilent Seahorse XFp T Cell Metabolic Profiling kit is designed to enable the acquisition ofrobust bioenergetic measurements of both glycolytic and mitochondrial activity in T cell andNK cell populations. These measurements can be associated with increased antitumor functionof cell therapy products. They are especially valuable in therapy development targeting to improvecell persistence and avoid exhaustion in a tumor microenvironment.1,2 The new kit allows you to
obtain more accurate measurements of the maximal respiratory capacity of T cells and NK cells
by using the uncoupler BAM15. BAM15 is less cytotoxic than other protonophore uncouplers,such as FCCP, and is especially suitable for sensitive cell types (such as naïve T cells and NK
cells). BAM15 also surpasses FCCP in its exhibition of a wider range of concentrations that induce
maximum respiration, minimizing the needs of uncoupler optimization.3
The kit supports multiple assay workflows.

XF T Cell Persistence assay
The Agilent Seahorse XF T Cell Persistence assay is developed to assess the ability of T cell
products (such as CAR-T) to develop durable immune response after infusion, and to generatememory-like T cells. Several publications indicate that T cells with a highly oxidative basal
metabolic phenotype (mitoATP/glycoATP production rate) and high spare respiratory capacity(SRC) exhibit increased persistence after adoptive transference.3 This assay can be used toevaluate different construct designs, engineering strategies, selection of starting material, or
metabolic conditioning during the manufacturing expansion process, to drive a metabolic
phenotype that correlates with persistent T cells. The typical assay profile and data output isshown in Figure 1.

Figure 1.

Typical Agilent Seahorse XF T Cell Persistence assay profile and data output.

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Introduction
XF T Cell Fitness assay

XF T Cell Fitness assay
The Agilent Seahorse XF T Cell Fitness assay is developed to evaluate if T cells can meet the
metabolic demand under altered or restrictive nutrient conditions. In this way, it assesses thecapacity of T cells to maintain metabolic fitness in a tumor microenvironment. The
nutrient-restrictive conditions can be created by using substrate pathway inhibitors (such as
etomoxir, UK5099, BPTES), by replacing glucose with other carbon sources, or by directly alteringlevels of metabolic substrates in the assay medium. In this assay, the basal ATP production rates
from both respiration and glycolysis (metabolic poise), as well as the maximal respiratory
capacity, are reported. The typical assay profile and data output are shown in Figure 2.

Figure 2.

Typical Agilent Seahorse XF T Cell Fitness assay profiles and data output.

XF NK Cell Metabolic Profiling assay
Cell metabolism and especially glycolysis are important drivers for NK cell anti-tumor function.
Using this kit, superior data and a comprehensive metabolic profile can be obtained for NK cells.
An injection prior to the kit reagents allows for evaluation of acute interventions, like fuel inhibitors
or metabolic modulators. The typical assay profile and data output is shown in Figure 3.

Figure 3.

Typical Agilent Seahorse XF NK Cell Metabolic Profiling assay.

Templates are provided on the XF T Cell Metabolic Profiling kit website. You can download thetemplates and upload them to your Agilent Seahorse XF HS Mini or XFp analyzer. Examples of
assay and controls groups, as well as instrument protocols, are shown in Figure 4.
6

Agilent Seahorse XFp T Cell Metabolic Profiling Kit User Guide

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Introduction
XF NK Cell Metabolic Profiling assay

Figure 4.

Template designs for assays using the Agilent Seahorse XFp T Cell Metabolic Profiling kit.

Data analysis and reporting results can easily be performed using Agilent Seahorse Analytics. A
valid assay kit is required for use of these analysis views. Otherwise, relevant analysis views will
not be accessible.
This user guide details how to use the XFp T Cell Metabolic Profiling kit with T cells and NK cells,
analyze assay data, and generate result reports.

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Introduction
Glossary

Glossary•

Compensatory glycolysis: The rate of glycolysis in cells following the addition of
mitochondrial inhibitors, driving compensatory changes in the cell to use glycolysis to meetthe cells' energy demands.

•

GlycoATP production rate: The rate of ATP production (expressed in pmol/min) associatedwith the conversion of glucose to lactate in the glycolytic pathway. For details of calculation,refer to the Agilent white paper by Romero et al.

•

Maximal respiration: The difference between the maximal OCR (obtained after exposure to an
uncoupler) and the OCR signal after Rot/AA addition. The uncoupler mimics a physiological“energy demand” by stimulating the respiratory chain to operate at maximum capacity, whichcauses rapid oxidation of substrates (sugars, fats, and amino acids) to meet this metabolic
challenge. It shows the maximum rate of respiration that the cell can achieve.

•

MitoATP production rate: The rate of ATP production (expressed in pmol/min) associatedwith oxidative phosphorylation in the mitochondria. It is calculated by multiplying the oxygen
consumption rate (OCR) signal between basal and oligomycin addition, with a P:O ratio. For
details of the calculation, refer to the Agilent white paper by Romero et al.

•

Percent of ATP from glycolysis (basal): The ratio of glycoATP and totalATP production rates.

•

Spare respiratory capacity: The difference between the maximal OCR signal (obtained after
exposure to an uncoupler), and the basal OCR signal. This measurement indicates thepotential (capacity) of a cell to respond to increased energetic demand, as well as how closely
the cell is respiring at its theoretical maximum. This parameter can be an indicator of cell
fitness or metabolic flexibility.

•

Spare respiratory capacity (%): The ratio of the maximal respiration, obtained with the signal
after exposure to an uncoupler and the signal after rotenone and antimycin A (Rot/AA)
addition; and the basal respiration, obtained with the basal OCR signal and the signal after
Rot/AA addition. The formula is: (OCRMax – OCRRot/AA) ÷ (OCRBasal – OCRRot/AA).

•

TotalATP production rate: The sum of mitochondrial and glycolytic ATP production rates inlive cells under a specific assay condition. This parameter represents the energy demand or
requirement for the cells under the conditions being tested.

References81

Kawalekar, O. U. Distinct Signaling of Coreceptors Regulates Specific Metabolism Pathways
and Impacts Memory Development in CAR T Cells. Immunity 2016, 44, 380–390.

2

Van Bruggen, J. A. C. et al. Chronic Lymphocytic Leukemia Cells Impair Mitochondrial Fitness
in CD8+ T Cells and Impede CAR T Cell Efficacy. Blood 2019, 134(1), 44–58.

3

Walls, J. et al. Assessing T Cell Bioenergetic Poise and Spare Respiratory Capacity Using
Extracellular Flux Analysis. Agilent Technologies application note, publication number:
5994-4494EN.

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Kit Information
Kit Contents 9
Kit Shipping and Storage 9
Additional Required Items 10

Kit Contents
The Seahorse XFp T Cell Metabolic Profiling kit includes six foil pouches, and each contains
reagents sufficient for a full-plate assay in an eight-well format. Every pouch includes one vial of
the following compounds: oligomycin A, BAM15, and a mixture of rotenone/antimycin A (seeTable 1).
Table 1

Agilent Seahorse XFp T Cell Metabolic Profiling kit contents.

CompoundFunction

Cap Color

Quantity Per Vial

Oligomycin A

ATP synthase inhibitor

Blue

6.75 nmol

BAM15

Mitochondrial membrane uncoupler

White

12.5 nmol

Rotenone/Antimycin A

Complex I and III inhibitor (respectively)

Red

2.75 nmol each

Kit Shipping and Storage
The XFp T Cell Metabolic Profiling kit can be shipped at ambient temperature and stored at room
temperature. The product is stable for 18 months from the date of manufacture. The actual
expiration date is printed on the label of the assay kit box. Depending on the shipping date, theactual shelf life of the kit in the user's hand can vary between 3 to 18 months.

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Kit Information
Additional Required Items

Additional Required Items
The products in Table 2 are also required for performing Seahorse XFp T Cell Metabolic Profilingassays, but are not supplied with the kit.
Table 2

Additional required items.

Item

Part number

Agilent Seahorse XF HS Mini analyzer or XFp analyzer
Agilent Seahorse XFp FluxPak (PDL miniplates)*

103721-100

Agilent Seahorse XF HS Mini FluxPak (PDL miniplates)*

103724-100

Agilent Seahorse XF RPMI Medium, pH 7.4†

103576-100

Agilent Seahorse XF 1.0 M Glucose Solution

103577-100

Agilent Seahorse XF 100 mM Pyruvate Solution

103578-100

Agilent Seahorse XF 200 mM Glutamine Solution

103579-100

Agilent Reservoir, 12 column, polypropylene orAgilent Reservoir, 12 column, polypropylene, irradiated201280-100 or
204365-100*

HS PDL miniplates are suitable for experiments with limited cells. XFp PDL miniplates are suitable for experiments with
abundant cell availability.

† Agilent Seahorse XF RPMI medium can also be purchased together with the supplements listed in this table as bundled
products (part number 103681-100).

10

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Assay Workflow for XF HS Mini and
XFp Analyzer
One Day Prior to Assay (Day 1) 12
Day of Assay (Day 2) 14
Data Analysis 19

Figure 5.

Agilent Seahorse XF T Cell Metabolic Profiling Kit with the Agilent Seahorse XF HS Mini and
XFp Analyzer.

Optimal cell seeding density should be empirically determined for your cell type prior to the assay.
Please refer to the “Frequently Asked Questions” section for recommendations. Additional
information can also be found on the Agilent Cell Analysis Learning Center website.
The Agilent Life Science Publication Database is an excellent resource for finding information
published in peer-reviewed research articles.

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Assay Workflow for XF HS Mini and XFp Analyzer
One Day Prior to Assay (Day 1)

One Day Prior to Assay (Day 1)
Power up the XF HS Mini or XFp Analyzer to allow the temperature to stabilize overnight(minimum of 18 hours).

Prepare the sensor cartridge and HS or XFp
PDL miniplate
1

Remove a three pack of the Agilent Seahorse Extracellular Flux assay kit from the box.
Remove the foil seal on the tub.

2

Place the sensor cartridge upside down next to the utility plate.

3

Fill each well of the utility plate with 200 µL of sterile, tissue culture-grade water.

4

Fill the moats around the wells (both sides) with water, using an eight-channel pipettor set to200 µL (Figure 6). This will result in a 400 µL volume in each chamber (two tips can fit into
each chamber). If no multichannel pipette is available, fill each chamber of the moat with
400 µL of water.

Figure 6.

N OTE

12

An eight-channel P200 pipettor is used to fill the moat on the utility plate.

5

Lower the sensor cartridge onto the utility plate, submerging the sensors in the calibrantor water.

6

Verify that the calibrant or water level is high enough to keep the sensors submerged.

7

Place the cartridge/utility plate assembly in a 37 °C, non-CO2 incubator overnight.

8

Transfer approximately 4 mL of calibrant solution to a 15 mL conical tube. Place it in a 37 °C,
non-CO2, nonhumidified incubator overnight.

9

Remove a precoated Agilent Seahorse XF HS or Seahorse PDL miniplate from the box andplace it in a 37 °C, non-CO2, nonhumidified incubator overnight to warm up the plate.

The XF HS PDL miniplate consists of a raised “ring” element in the center of each well, and ispre-assembled with a silicone cell-seeding mask. This allows performance of XF assays withsignificantly fewer cells per well, facilitating functional analysis of non-proliferative or limited
amounts of cells. Please review the document Seeding Suspension Cells in Agilent Seahorse
XF HS PDL Miniplates, prior to performing any XFp assays using the XF HS PDL miniplate, fordetailed information about this miniplate.

Agilent Seahorse XFp T Cell Metabolic Profiling Kit User Guide

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Assay Workflow for XF HS Mini and XFp Analyzer
Design and upload the assay template to the XF HS Mini or XFp analyzer

Design and upload the assay template to the XF HS Mini
or XFp analyzer
1

On a computer with internet access, download the default template(s) on the XF T Cell
Metabolic Profiling Kit website.

2

Unzip the folder to retrieve the templates. If necessary, you may modify the templates using
Wave Desktop.

3

Transfer the templates to a USB drive. Templates must reside on the root of the USB flash
drive and not in a subfolder.

4

Insert the USB drive into the front USB port on the Seahorse XF HS Mini or XFp analyzer.

5

Click Settings, then click Template Management.

6

Click the USB tab, and locate the assay template(s) to upload.

7

Select the check box next to each assay template to be imported.

8

Click Import.

9

All imported assay templates are now available for selection from the Local tab.

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Assay Workflow for XF HS Mini and XFp Analyzer
Day of Assay (Day 2)

Day of Assay (Day 2)
Prepare the cartridge
Complete hydration of the sensor cartridge by removing the water and adding 200 µL of
prewarmed calibrant to each well in the utility plate. Incubate in a 37 °C, non-CO2 incubator for
60 minutes.

Prepare the assay medium
1

Prepare standard Seahorse XF RPMI assay medium by supplementing 19.4 mL of Seahorse
XF RPMI Medium, pH 7.4 with 0.2 mL each of Agilent Seahorse XF glucose, pyruvate, andglutamine solutions (10 mM, 1 mM, and 2 mM final concentrations in assay medium,
respectively), as shown in Table 3. These are the recommended initial substrate conditions.
However, the desired assay medium composition can be modified depending on the userneeds.

Table 3Component

Volume (mL)

Final Concentration (mM)

XF RPMI Medium, pH 7.4

19.4

–

XF 1.0 M Glucose Solution

0.2

10.0

XF 100 mM Pyruvate Solution

0.2

1.0

XF 200 mM Glutamine Solution

0.2

2.0

2

N OTE

14

Agilent Seahorse XF RPMI assay medium.

Warm up to 37 °C.

No pH adjustment to the assay medium is necessary when nonsaturating concentrations of
substrates, listed in Table 3, are used for XF assay medium.

Agilent Seahorse XFp T Cell Metabolic Profiling Kit User Guide

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Assay Workflow for XF HS Mini and XFp Analyzer
Prepare and seed cells in the Seahorse HS or XFp PDL miniplates

Prepare and seed cells in the Seahorse HS or XFp
PDL miniplates
Important: The procedure described below is also known as the Seahorse XFp assay suspension
cell protocol. Before starting any experiments, please read the information in the “Frequently
Asked Questions” section, which contains information about the best practices for handling cells,
counting cells, and normalizing data, to ensure that optimal results are achieved.
1

Take a small sample of cell suspension and count the cells.

2

Calculate the total number of cells needed (desired number of cells per well multiplied by the
number of wells to be seeded).
For example: 100,000 cells per well × 6 wells = 6.0 × 105 cells.

N OTE

N OTE

It is recommended to allow at least 50% extra cells to account for the potential loss of cells
during centrifugation and other steps.
3

Transfer the appropriate volume of cell suspension from the culture vessel to a conical tube.
Preferentially, use 15 mL conical tubes for best cell recovery. Use multiple tubes if needed,
then pool the cells after resuspending them following centrifugation.

4

Centrifuge the cells at room temperature at 200 ×g for 10 minutes. Calculate the volume
required to resuspend the cell pellet. We recommend using a volume 20% smaller than thecalculated volume to avoid over-diluting the sample.

5

Remove the supernatant carefully from the centrifuged conical tube using a pipette (do not
aspirate, to minimize cell loss).

6

Resuspend the cells in warm assay medium. Take a small sample and perform the cellcounting again. Adjust the volume to achieve the desired cell concentration as indicated inTable 4, for different types of cells and miniplates. In general, the XF HS PDL miniplate willusually accommodate one-third the number of cells that an XFp PDL miniplate requires. See
Using the Agilent NovoCyte Flow Cytometer for Immune Suspension Cell Normalization in
Agilent Seahorse XF Assays for more detailed information.

Table 4 lists the potential starting cell densities as tested in Agilent laboratories. It is recommended to perform density optimization when you start working with your cells to ensure optimal results (also see the “Frequently Asked Questions” section).
Table 4

Starting cell seeding conditions for the different types of cells and plates.

Plate Type

Cell Type

Cell Suspension(Total Cells/mL)

Seeding Volume(µL/Well)

Final Cell Density(Total Cells/Well)

HS PDL MiniplateNaïve T Cell

1.7 × 106 to 2.3 × 106

30

5.0 × 104 to 7.0 × 104

Activated T Cell

6.7 × 105 to 1.2 × 106

30

2.0 × 104 to 3.5 × 104

NK Cell

1.7 × 106 to 2.7 × 106

30

5.0 × 104 to 8.0 × 104

Stimulated NK Cell

6.7 × 105 to 1.2 × 106

30

2.0 × 104 to 3.5 × 104

Naïve T Cell

3.0 × 106 to 4.0 × 106

50

1.5 × 105 to 2.0 × 105

Activated T Cell

1.2 × 106 to 2.0× 106

50

6.0 × 104 to 1.0 × 105

NK Cell

3.0 × 106 to 5.0× 106

50

1.5 × 105 to 2.5 × 105

Stimulated NK Cell

1.0 × 106 to 2.0× 106

50

5.0 × 104 to 1.0 × 105

XFp PDL Plate

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Assay Workflow for XF HS Mini and XFp Analyzer
Prepare and seed cells in the Seahorse HS or XFp PDL miniplates
7

Remove the warm XF HS PDL miniplate or XFp PDL miniplate from the incubator.

8

Pipette 30 µL or 50 µL of cell suspension to wells B through G for XF HS or XFp PDL
miniplates, respectively, using a single channel pipettor. The pipette tip must be placed at thebottom of the well and in the inner ring element of the wells on the XF HS PDL miniplate, todispense properly (Figure 7). Do not add cells to wells A and H.

Figure 7.

9

The pipette tip (outlined in blue) is inserted to the bottom of each well to dispense cellsuspension.

Add 30 µL or 50 µL of assay medium to wells A and H (background wells) for XF HS or XFp
PDL miniplates, respectively.

10 Fill the moats around the wells (both sides) with warm water or PBS, using an eight-channel
pipettor set to 200 µL, as demonstrated in Figure 5.

N OTE

Seeding volume is critical for achieving a uniform distribution of cells within a well. Do notseed cells in a volume higher than what is recommended for each plate type before thecentrifugation step (See Table 4).
11 Centrifuge gently (e.g., 200 ×g for 1 minute) to allow the cells to attach to the bottom of thewells. It is recommended to use a centrifuge break.
12 If XF HS PDL miniplate is used, follow the steps described in the document Seeding
Suspension Cells in Agilent Seahorse XF HS PDL Miniplates to remove the silicone mask.
13 The final number of cells per well varies depending on the types of miniplates and cells used.
(See Table 4.)
14 Gently add additional warm XF assay medium to achieve the optimal total starting volume,
according to Table 5, which varies for different assay and plate types.
Table 5

Standard cell seeding conditions for the different types of T cells and plates.
Additional Assay
Medium Volume(µL/Well)

Total Starting
Volume(µL/Well)Assay Type

Plate Type

Seeding Volume(µL/Well)Assay Without an
Acute Injection

HS PDL miniplate

30

170

200

XFp PDL miniplate

50

150

200

Assay with an Acute
Injection

HS PDL miniplate

30

145

175

XFp PDL miniplate

50

125

175

15 Incubate the miniplate at 37 °C in a non-CO2 incubator for 45 to 60 minutes prior to the assay.
16

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Assay Workflow for XF HS Mini and XFp Analyzer
Prepare the compound solutions and load the injection ports

Prepare the compound solutions and load theinjection ports
Important: It is not necessary to perform titration experiments for the compounds provided in the
kit when T cells or NK cells are used.This is based upon testing with a panel of NK cell and T cell
types.3 Check the “Frequently Asked Questions” section for more guideline on working with othertypes of cells. Use the compounds on the same day that they are reconstituted. Discard any
remaining compound solutions. Do not refreeze and reuse.
1

Remove one foil pouch from the Agilent Seahorse XFp T Cell Metabolic Profiling kit box.

2

Open the pouch. Remove all three vials with a gloved hand.

3

Add 0.5 mL of XF assay medium to each vial to resuspend the content (see Table 6). Vortex
for 30 seconds, if needed, to ensure that the content dissolves completely.

Table 6Compound solutions.

CompoundCap Color

Volume to Add (mL)

Solution Concentration (µM)

Oligomycin A

Blue

0.5

13.5

Bam15

White

0.5

25

Rotenone/Antimycin A

Red

0.5

5.5

4

Load 25 µL of compound to each injection port, according to Table 7. For the location of theports, refer to Figure 8.

Table 7

Assay type, injection port, and volume recommendations for eight-well miniplates.

CompoundPort
Concentration

Assay Without
Acute Injection
Port

Assay With an
Acute Injection

Loading
VolumePort

200 µL

Loading
VolumeFinal
Concentration

Starting
Volume–175 µL–

Test
Compound

8X––

Port A

25 µL

1X

Oligomycin A

9X

Port A

25 µL

Port B25 µL

1.5 µM

BAM15

10 X

Port B

25 µL

Port C25 µL

2.5 µM

Rotenone/
Antimycin A

11 X

Port C

25 µL

Port D25 µL

0.5 µM each

.

N OTE

Proper port-loading techniques can be found in Basic Procedure “Loading the Sensor
Cartridge with Compounds”, on the Agilent Cell Analysis Learning Center website. Please readthe information prior to loading compounds. Ensure that the sensor cartridge is properly
hydrated prior to use.

Figure 8.

Location and numbering of injection ports on Agilent Seahorse XF sensor cartridges.

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Assay Workflow for XF HS Mini and XFp Analyzer
Running the assay on the XF HS Mini analyzer or XFp analyzer

Running the assay on the XF HS Mini analyzer or XFp
analyzer
N OTE

18

If your assay template is not uploaded to the XF HS Mini or XFp analyzer, refer to section“Design and upload the assay template to the XF HS Mini or XFp analyzer” on page 13 to
upload the assay template.
1

On the XF HS Mini analyzer or XFp analyzer Home view, click Start to display the list ofavailable assay templates.

2

Select the assay template from the list of available templates on the Local tab. Click to open
the template.

3

Review groups (cell types or conditions), plate layout map, and instrument protocol by using
the menu on top of the XF HS Mini analyzer display, or by touching the arrow on the lower rightcorner on XFp analyzer display.

4

You can edit the assay result file name, add notes about the assay, and review alert
notifications on the review and run display on XF HS Mini analyzer, or by clicking the arrow on
the lower right corner on XFp analyzer display. You can also enter an email address to receive
automated notifications from your analyzer when user interaction is required, and a copy of
the assay result file when the assay has completed. An active internet connection is required
for this feature.

5

Click Start Run when ready to start the assay.

6

When prompted, remove the cartridge lid and place the loaded sensor cartridge with the utility
plate on the thermal tray on the analyzer. Ensure correct plate orientation and that the
cartridge lid has been removed. Then, click I'm Ready. Calibration takes approximately 15 to
20 minutes.

7

After completing calibration, the screen will display the Load Cell Plate dialog. Click Open Trayto eject the utility plate and load the cell plate. Ensure that the lid is removed from the cell plate
before loading.

8

Click Continue to run the assay.

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Assay Workflow for XF HS Mini and XFp Analyzer
Data Analysis

Data Analysis
Seahorse Analytics is a web-based software platform that provides a simple, streamlined data
analysis workflow.
After obtaining assay result files, it is recommended to perform a data quality review with either
Wave Desktop software or in Seahorse Analytics, before moving further to analyzing the data. A
data quality review is performed by manually examining the O2 and pH level data for each well.
Outliers, if any, can be excluded at this step.
Follow the steps below to use the relevant assay widgets in Seahorse Analytics:
1

After the assay is completed, transfer your assay result file to your office or personal computer
using a USB drive or network drive.

2

To register or log in to your Seahorse Analytics account, visit:https://seahorseanalytics.agilent.com.

3

Import the assay result file to your account.

4

Click the three-dot menu to the right of the file on the Files view (only). Click Add Assay Kit.

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Assay Workflow for XF HS Mini and XFp Analyzer
Data Analysis

5

In the pop-up dialog box, enter assay kit information (kit part number, lot number, and SW ID).
Click Validate. This unlocks the assay analysis views.

6

Click the result file to display the Add View window. If a data quality review has not been
performed, it can be done by using the Standard Graph widgets. Outliers, if any, can be
excluded at this step using the Modify function. For more information about this function,please visit the Agilent Cell Analysis Learning Center.

7

After the data quality review, open the Add View window again. Click the XF T Cell Persistence
Assay analysis view, the XF T Cell Fitness Assay analysis view, or the XF NK Cell Metabolic
Profile analysis view.

8

Select the desired analysis view(s) in the View Widgets window, then click Add View.

For additional information about using Seahorse Analytics, visit the Agilent Cell Analysis Learning
Center website.

20

Agilent Seahorse XFp T Cell Metabolic Profiling Kit User Guide

4

Frequently Asked Questions
How should T cells be handled to maximize the chance of a successful assay?
Cell preparation is one of the most critical steps to ensure consistent and robust response. Hereare some tips for you to consider:-

Never put your cells on ice and never spin cells in a cold centrifugation chamber. Metabolism
is strongly affected by temperature changes. In general, lowered temperature will slow or altermetabolism and exert stress on cells, leading to reduced metabolic rates and higher
assay variation.

-

Warm up the PDL cell culture plate overnight prior to use. Plastic plates can absorb O2 fromthe air. When the temperature increases, the O2 absorbed in the plates is slowly released into
the wells. This process can take hours to reach equilibrium. If cells are plated onto a cold PDL
plate, the O2 in the plate is then slowly released into the assay medium during the assay. Thiscan interfere with assay signals, especially in the case when the OCR signal is in the low
detection range.

-

Ensure consistent cell counting by counting both total cells and viable cells. This is critical forachieving consistent assay results. Ensure that you obtain both the total cell number, as wellas the viable cell number (or a percentage). See Using the Agilent NovoCyte Flow Cytometer
for Immune Suspension Cell Normalization in Agilent Seahorse XF Assays for more detailed
information.

-

Count cells twice: before centrifugation, and after resuspending cells in assay medium. The
second counting in step 5 in Figure 9 is especially important to ensure accurate seeding
density, and should not be omitted because some cells can be lost when discarding
spent medium.

Figure 9.

-

Suggested cell-preparation steps for cell seeding with T cells.

Use total cell number (live + dead cells) to calculate the number of cells needed for each well.
Both live and dead cells occupy space at the bottom of wells. Therefore, you should use total
cell count to calculate medium volume and dilution factor for cell seeding, not the viable cellcount. This is especially important when you have a sample with less than 90% viable cells. In
this case, if you only consider viable cells, you may end up seeding too many cells per well,leading to high assay variability. Viable cell count is only useful in performing normalization of
signals.

Agilent Seahorse XFp T Cell Metabolic Profiling Kit User Guide

21

4Frequently Asked Questions-

Minimize the number of centrifugation steps during cell preparation. We recommend spinningcells only once during cell preparation. This means performing cell counting first to obtain the
total number of cells you have, before spinning the cells. This will give you an idea about thevolume of assay medium you will need to resuspend the pellet without over-diluting your cellsuspension; avoiding spinning the cells again.

Can I thaw frozen cells and seed directly to XF PDL culture plates to perform XF assays on thesame day?
When working with frozen cells, it is recommended to thaw the cells in cell maintenance medium
overnight before using the cells to perform XF assays. This is because cellular metabolism takes
time to recover. The overall signal can be low if overnight recovery is not implemented. If it isdesired to obtain data from the cells right after thawing, it is recommended to perform a second
assay with the same batch of cells after recovering in cell maintenance medium overnight.
Do you provide a tool for calculating the cells required at each of the cell-preparation steps?
We do not provide a tool for this purpose. However, you can use Table 8 as an example of how to
calculate the number of cells you will need for your assay.
Table 8

Example calculations for preparing a cell suspension to seed six wells in an
Agilent Seahorse XF HS PDL miniplate.

Desired seeding density and volume per well

3.0 × 104 cells in 30 µL

Total number of cells needed for an experiment with
30 wells plus 50% extra(3.0 × 104 cells × 6 wells) × 1.5 = 2.7 × 105 cells

Target cell density for the final cell suspension in
assay medium (to be used in seeding plate)

3.0 × 104 cells / 30 µL = 1.0 × 106 cells/mL

Cell count for cell suspension in growth medium(total cells)

1.5 × 106 cells/mL

Amount of cell suspension needed

1.0 × 106 cells / 1.5 × 106 cells/mL = 0.67 mL

Spin and carefully remove cell culture medium
Recommended assay medium volume to resuspend cellpellet (= 70% of the theoretical volume assuming there is
no loss of cells)(1.0 × 106 cells) / 1.0 × 106 cells/mL × 70% = 0.7 mL

Cell count for cell suspension in assay medium(use 100 µL aliquot, dilute if needed)

1.3 × 106 cells/mL

Cell viability

92%

Total cells (live + dead) in 0.6 mL cell suspension (amountleft after 100 µL is used for cell counting)(1.3 × 106 cells/mL) × 0.6 mL = 0.78 × 106 cells

Additional assay medium needed to achieve target celldensity for the final cell suspension(0.78 × 106 cells / 1.0 × 106 cells) - 0.6 mL = 0.18 mL

Transfer 50 µL to each well

22

Total cells per well (live + dead)

3.0 × 104 cells

Cell number to use for normalization(3.0 × 104 cells) × 0.92 = 2.76 × 104 cells/well

Agilent Seahorse XFp T Cell Metabolic Profiling Kit User Guide

4

Frequently Asked Questions

Do I need to perform a cell density optimization experiment, and how?
Yes. We recommend a test, such as microscopic evaluation of the monolayer, to optimize
cell-plating density when you first start performing XF assays. The seeding densities
recommended in Table 4 serve only as a reference point, and you should plan testing with acouple of seeding densities above and below the recommended numbers. This is because
different cell-counting methods used by different users or in different labs may yield different
counting results, which could lead to suboptimal seeding density on the XF cell culture
microplates. If cell density is below optimal, it may yield reduced signal level. In contrast, if a well isoverloaded with cells, some of the cells cannot attach to the bottom of the well and can be pushedaside during the assay when compounds are injected. This can result in high well-to-well variationin assay results. In general, cells should be distributed evenly as a monolayer at the bottom of theplate after the centrifugation step, with some space between the cells (see Figure 10). The
optimal cell density after the centrifugation step should show a cell distribution similar to
Figure 10A, or slightly higher, and should never be more than 100% confluent (not denser thanwhat is shown in Figure 10B).

Figure 10.

Microscopic view of optimal cell seeding densities: (A) Low end of optimal cell density. (B) Highend of optimal seeding density.

Do I need to perform experiments to optimize the concentrations of injection compounds fordifferent types of T cells?
Extensive tests have been performed with different types of human T cells and NK cells fromseparate donors, as well as mouse T cells and NK cells. The results show that the recommended
compound concentrations work well for all T cell types and NK cell types. Therefore, it is notnecessary to perform concentration optimization experiments when working with T cells or
NK cells.
Can I use the XF T Cell Metabolic Profiling kit for other types of cells (non-T cells or NK cells)?
In theory, the kit should work for all types of cells. However, verification and optimization
experiments (such as seeding density and modulator concentration) are required to use the kit
with non-T cells. Table 9 shows a suggested concentration range for preparing reagents, toperform a concentration optimization experiment.. For information on how to optimize seeding
density, refer to the Agilent Seahorse XF Learning Center.

Agilent Seahorse XFp T Cell Metabolic Profiling Kit User Guide

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4Frequently Asked Questions

Table 9

Recommended concentration range for injection compound optimization experiments.

Compounds

Oligomycin A

BAM15

Rotenone/
Antimycin A

Assay Medium
per Vial (mL)

250

416

250

Stock
Concentration
(µM)

27

30

11

Loading Solution

Fold in Port

Final
Concentration(µM)

Stock Solution
(µL)

Assay
Medium (µL)

Concentration
(µM)

40

80

4.5

60

60

13.5

80

40

18.0

2.0

40

80

10.0

1.0

60

60

15.0

1.5

80

40

20.0

100

20

25.0

2.5

120

0

30.0

3.0

30

90

2.75

0.25

60

60

5.5

90

30

8.25

1.0
9x

10x

11x

1.5

2.0

0.5
0.75

Why is it important to ensure an optimal seeding density? How can I normalize my suspension
cell data between experiments?
To obtain robust XF data, suspension cells need to be seeded in an optimal density range. This
range must produce measurements within the recommended range of the instrument, and also
provide an even distribution of the cells on the surface of the cell culture plate. Lower than optimal
seeding densities may yield a signal that is below the limit of detection of the instrument, while
higher than optimal seeding densities can result in increased well-to-well variation of the assay
results. Using the Agilent NovoCyte Flow Cytometer to count live cells at several points during thecell seeding workflow allows for optimal seeding and for normalization of XF data between
experiments.
See Using the Agilent NovoCyte Flow Cytometer for Immune Suspension Cell Normalization in
Agilent Seahorse XF Assays for more detailed information.
I cannot access Seahorse Analytics. What should I do?
If you experience any issues using Seahorse Analytics, please contact the Agilent Cell Analysis
Support team, at: cellanalysis.support@agilent.com, to report your issue. You should also
double-check your internet connection and/or web browser. Seahorse Analytics works best with
Google Chrome, Safari (Apple), Mozilla Firefox, and Microsoft Edge. For any other issues
preventing you from using Seahorse Analytics for data analysis, you can export your data to
Microsoft Excel and GraphPad Prism to quantify and plot maximum responses and area under
the curve data. For more information on how assay parameters are calculated, refer to the Agilent
application note, 5994-4494EN.3

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Agilent Seahorse XFp T Cell Metabolic Profiling Kit User Guide

www.agilent.com
For Research Use Only. Not for use in diagnostic
procedures.
RA44560.4251041667

Agilent Technologies, Inc. 2024
Second edition, March 2024

5994-3494EN Rev. C