Agilent Seahorse XFp
Mito Fuel Flex Test KitFor use with Agilent Seahorse
XFp Extracellular Flux Analyzers

User Manual 
Kit 103270-100

Agilent Technologies

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Manual Part Number
103270-400

Kit Part Number
103270-100

EditionSecond edition, April 2019
Revision F0
Printed in USA
Agilent Technologies, Inc.
2850 Centerville Road Wilmington, DE 19808-1610 USA

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Contents
Introduction
Assay Background
Glossary

5

10

Kit Information
Kit Contents

11

Kit Shipping and Storage

11

Additional Required Items

12

Assay Workflow
Day Prior to Assay
Day of Assay

14

Running the Assay
Data Analysis

14

19

19

Frequently Asked Questions

Agilent Seahorse XFp Mito Fuel Flex Test Kit User Manual3

4

Agilent Seahorse XFp Mito Fuel Flex Test Kit User Manual

Agilent Seahorse XFp Mito Fuel Flex Test Kit
User Manual

1
Introduction
Assay Background 5
Glossary 10

Assay Background
The Agilent Seahorse XF Mito Fuel Flex Test is a method formeasuring mitochondrial fuel usage in live cells. In combination
with the Agilent Seahorse XFp Analyzer, the Agilent Seahorse
XF Mito Fuel Flex Test Kit measures the dependency, capacity,and flexibility of cells to oxidize three mitochondrial fuels:• Glucose (pyruvate)• Glutamine (glutamate)• Long-chain fatty acids.
The Seahorse XF Mito Fuel Flex Test determines the rate ofoxidation of each fuel by measuring mitochondrial respiration[the oxygen consumption rate, OCR] of cells in the presence orabsence of fuel pathway inhibitors (Figure 1 on page 6).
Sequentially inhibiting the pathway of interest followed by thetwo alternative pathways enables the calculation of how
dependent the cells are on the pathway of interest to meet basal
energy demand (Figure 2 on page 7). Dependency indicates thatthe cells’ mitochondria are unable to compensate for theblocked pathway by oxidizing other fuels. Inhibiting the twoalternative pathways followed by the pathway of interest
enables the calculation of cells' mitochondrial capacity to meetenergy demand (Figure 3 on page 8). Fuel Flexibility is
calculated by subtracting the Fuel Dependency from the Fuel
Capacity for the pathway of interest (Figure 4 on page 9).
Flexibility indicates the cells’ mitochondria have the ability tocompensate for the inhibited pathway by utilizing otherpathways to fuel mitochondrial respiration. The presence ofdependency and absence of flexibility demonstrates that the
mitochondria require that fuel pathway to maintain basal OCR.
The Seahorse XF Mito Fuel Flex Test Kit contains three pathway
inhibitors required to determine the dependency, capacity, andflexibility of cells for glucose, glutamine and long chain fatty
acids.

Agilent Technologies

5Introduction• UK5099 - An inhibitor of the glucose oxidation pathway.
UK5099 blocks the mitochondrial pyruvate carrier (MPC).
Cells convert glucose to pyruvate through glycolysis.
Pyruvate can be transported into the mitochondria andoxidized by the TCA cycle.
• BPTES - An inhibitor of the glutamine oxidation pathway.
BPTES is an allosteric inhibitor of glutaminase (GLS1).
Glutaminase converts glutamine to glutamate, glutamate isthen converted to alpha-ketoglutarate and oxidized by the
TCA cycle.
• Etomoxir - An inhibitor of long chain fatty acid oxidation.
Etomoxir inhibits carnitine palmitoyl-transferase 1A(CPT1A), which is critical for translocating long chain fatty
acids from the cytosol into the mitochondria for betaoxidation.

Figure 1

6

Principle of the Agilent Seahorse XF Mito Furl Flex Test
Energy produced by cells can be derived from mitochondrial
oxidation of glucose, glutamine, and fatty acids. The cells’
mitochondrial dependency on and flexibility for each of thesefuel sources is determined by measuring the decrease in fueloxidation (decline in oxygen consumption rate) upon additionof one or more inhibitors.

Agilent Seahorse XFp Mito Fuel Flex Test Kit User ManualIntroduction

Figure 2

Fuel Dependency: Glutamine Oxidation Pathway Example
Fuel Dependency is tested by first injecting an inhibitor of thetarget pathway, followed by inhibition of the two alternative
pathways. Dependency is calculated using the equation
shown here. In this example, HepG2 cells were tested for
Glutamine Pathway Dependency (n=3). BPTES was injectedfollowing the third rate measurement (“Baseline”
measurement). The sixth rate measurement following thisinjection is used for the calculation to allow sufficient time forthe compound to have a complete effect (“Target Inhibitor”
measurement). The final rate measurement (“All Inhibitors”) isused to complete the equation.

Agilent Seahorse XFp Mito Fuel Flex Test Kit User Manual7

Introduction

Figure 3

8

Fuel Capacity: Glutamine Oxidation Pathway Example
Fuel Capacity is tested by first injecting inhibitors of thealternative pathways, followed by inhibition of the target
pathway. Dependency is calculated using the equation shown
here. In this example, HepG2 cells were tested for Glutamine
Pathway Capacity (n=3). Eto and UK5099 were injectedfollowing the third rate measurement (“Baseline”
measurement). The sixth rate measurement following thisinjection is used for the calculation to allow sufficient time forthe compounds to have a complete effect (“Other 2 Inhibitors”
measurement). The final rate measurement (“All Inhibitors”) isused to complete the equation.

Agilent Seahorse XFp Mito Fuel Flex Test Kit User ManualIntroduction

Flexibility% = Capacity% - Dependency%

Figure 4

Fuel Flexibility: Glutamine Oxidation Pathway Example
Determination of Flexibility requires two groups: one
Dependency group and one Capacity group (Figure 2 on page 8
and Figure 3 on page 9). Fuel Flexibility is calculated as thedifference between Capacity and Dependency. All threeparameters (Dependency, Capacity, and Flexibility) aredisplayed as a stacked bar chart when using the Agilent
Seahorse XF Mito Fuel Flex Test Report Generator. See “DataAnalysis” on page 19 for further information.

Agilent Seahorse XFp Mito Fuel Flex Test Kit User Manual9Introduction

Glossary
Baseline Respiration Rate of oxygen consumption due to fueloxidation under initial assay conditions.
Fuel A substrate or nutrient that is used by cells and oxidizedin the mitochondria. In this assay, the mitochondrial oxidationof glucose, glutamine, and/or long chain fatty acids is measured.
Fuel Pathway A series of biochemical processes that convert
fuels into metabolites that are oxidized in the mitochondria(example: the conversion of glucose to pyruvate and transportof pyruvate into mitochondria).
Fuel Dependency The measurement of cells' reliance on aparticular fuel pathway to maintain baseline respiration.
Fuel Capacity The ability of a cell's mitochondria to oxidize afuel when other fuel pathways are inhibited.
Fuel Flexibility The difference between fuel capacity anddependency, that is, the ability of cells to increase oxidation of aparticular fuel in order to compensate for inhibition ofalternative fuel pathway(s).

10Agilent Seahorse XFp Mito Fuel Flex Test Kit User Manual

Agilent Seahorse XFp Mito Fuel Flex Test Kit
User Manual2
Kit Information
Kit Contents 11
Kit Shipping and Storage 11
Additional Required Items 12

Kit Contents
The Agilent Seahorse XFp Mito Fuel Flex Test Kit contains
sufficient compounds to complete six fuel dependency or
combined dependency and flexibility tests (Figure 5 onpage 13). The kit includes six foil pouches, each pouch contains
one tube of each of the following compounds.
Table 1

Kit compounds

CompoundCap color

Amount per tube (nmol)

BPTES

Grey

6

EtomoxirGreen

8

UK5099Orange4

Kit Shipping and Storage
Product ships at ambient temperature. Product can be stored atroom temperature and is stable for one year from the date of
manufacture. The expiration date is printed on the label of the
kit box. Depending on the shipping date, the actual shelf life of
the product in users' hand varies from 12 to 3 months.

Agilent Technologies

11Kit Information

Additional Required Items
The following items are also required for performing Seahorse
XF Mito Fuel Flex Tests, but they are not supplied with the kits.
Table 2

Additional required items

Item

Supplier

Catalog number

Agilent Seahorse XFp Analyzers

Agilent Technologies

Seahorse XFp FluxPak(cartridges, miniplates, andcalibrant)

Agilent Technologies

103022-100

XF DMEM medium, pH 7.4*
or
XF RPMI medium, pH 7.4*

Agilent Technologies

103575-100
103576-100

XF 1.0 M Glucose solution

Agilent Technologies

103577-100

XF 100 mM Pyruvate solution

Agilent Technologies

103578-100

XF 200 mM Glutamine solution

Agilent Technologies

103579-100

Microfuge tubes

Various0.5-1.5 capacity* XF DMEM or RPMI media can also be purchased together with the supplements listed inthis table as bundled products (Catalog Number 103680-100 and 103681-100). For a full listof all medium types and our recommendation for each assay kit, please refer to the
Seahorse XF Media Selection Guide.
http://www.agilent.com/cs/library/selectionguide/public/5991-7878EN.pdf

12

Agilent Seahorse XFp Mito Fuel Flex Test Kit User Manual

Agilent Seahorse XFp Mito Fuel Flex Test Kit
User Manual

3
Assay Workflow
Day Prior to Assay 14
Day of Assay 14
Running the Assay 19
Data Analysis 19

Figure 5

Seahorse XF Mito Fuel Flex Test Assay Workflow for the Agilent Seahorse XFp Analyzers

Agilent Technologies

13

Assay Workflow

Day Prior to Assay
1

For adherent cells, plate cells at a previously determined
density in the XFp Cell Culture Miniplate using theappropriate cell culture growth medium, adding medium
only to wells A and H as well as the moats. Refer to Basic
Procedures for the Agilent Seahorse XFp Analyzer:
https://www.agilent.com/en/products/cell-analysis/how-torun-an-assay

2

Hydrate a sensor cartridge in Agilent Seahorse XF Calibrantat 37 °C in a non-CO2 incubator overnight. Refer to Basic
Procedures for the Agilent Seahorse XFp Analyzer.

3

Download the Seahorse XF Mito Fuel Flex Report Generator
and both Dependency and Flexibility Assay Templates fromthe Agilent.com website. Seahorse recommends using the
Assay Templates provided to perform the Dependency or
Flexibility workflow. Both Assay Templates can be modified
or customized using Wave Desktop.
https://www.agilent.com/en/products/cell-analysis/cell-analysis-software/data-analysis/seahorse-xf-mito-fuel-flex-test-r
eport-generator
The Dependency or Flexibility assay may also be set up
directly on the Agilent Seahorse XFp Analyzer. First, selectthe Blank Template then, after each injection, adjust the
number of cycles to 6. See the Agilent Seahorse XFp
Extracellular Flux Analyzer User Guide for additional
details.

Day of Assay

14

1

Turn on the Agilent Seahorse XFp Analyzer and let it warmup (minimum 20 minutes).

2

Determine which type of assay you will run (dependency or
flexibility), and which fuel pathway you will test (glucose,glutamine, or fatty acid). One Agilent Seahorse XFp
Miniplate can accommodate one fuel assay: either apairwise comparison of fuel dependency, or a single cellcondition with both dependency and flexibility.

Agilent Seahorse XFp Mito Fuel Flex Test Kit User Manual

Assay Workflow

Prepare Assay medium
1

Prepare assay medium by supplementing Seahorse XF
DMEM or RPMI medium. It is recommended to start with
1 mM pyruvate, 2 mM glutamine, and 10 mM glucose.
However, medium composition can be changed depending
on cell type or the desired study conditions. For more
information, refer to the Basic Procedure, “Preparing Assay
medium for Use in XF Assays”, on the Agilent Cell Analysis
Learning Center.
www.agilent.com/en/products/cell-analysis/how-to-run-anassay

2

Bring XF medium with pH 7.4 and XF supplements into acell culture hood. Transfer a sufficient volume of XF
medium to a sterile bottle. It is not necessary to warm themedium and supplement before this step.

3

Add proper volumes of XF supplements to achieve thedesired final concentrations. This is your assay medium.
When recommended supplement concentrations are used,
pH adjustment is not necessary.

4Warm the assay medium to 37 °C in a water bath. It is readyto use.

Prepare Agilent Seahorse XFp Cell Culture Miniplate for Assay• Adherent cells
1 Remove cell culture miniplate from 37 °C CO2 incubatorand examine cells under microscope to confirm that cells
are 50-90 % confluent and evenly distributed within eachwell.
2 Remove assay medium from water bath.
3 Remove the cell culture growth medium in the cell

culture miniplate and wash with warm assay medium.
Remove the wash and add assay medium to total volumeof 180 µL. Place the cell culture miniplate into a 37 °C
non-CO2 incubator for 1 hour prior to the assay.
• Suspension cells
1 Harvest, count, and resuspend cells in assay medium atthe desired density.
2 Add 50 µL assay medium without cells to wells A and H

of an Agilent Seahorse XFp Cell Culture Miniplate.
3 Add 50 µL cells suspended in assay medium to wells

B through G.

Agilent Seahorse XFp Mito Fuel Flex Test Kit User Manual

15

Assay Workflow

4 Centrifuge miniplate in Agilent Seahorse XFp Carrier

Tray at 300 × g for 1 minute.
5 Add 130 µL medium to each well to a total volume of

180 µL.
6 Place the miniplate into a 37 °C non-CO2 incubator for

45 minutes prior to the assay.

Load template onto the Agilent Seahorse XFp Analyzer(If template(s) already present, skip this step.)
1

Visit the Seahorse Bioscience website, and download theAgilent Seahorse XF Mito Fuel Flex Report Generator alongwith both Dependency and Flexibility Assay Templates.
Both Seahorse XFp Mito Fuel Flex Test - Dependency andSeahorse XFp Mito Fuel Flex Test - Flexibility Assay
Templates are included in the downloaded folder.

2

Copy the unzipped Assay Templates (*.asyt) files to the root
directory of a USB Flash Drive.

3

Insert the USB drive into the front USB port of the Seahorse
XFp Analyzer.

4

Click Settings, then click Template Management.

5

Click the USB tab, and locate the Assay Template(s) to
upload.

6

Check the checkbox next to each Assay Template to beimported, then click Import.

7

All imported Assay Templates will now be available forselection from the Local tab when starting an XFp assay.

Removing reagent caps
Hold tube in gloved hand and roll thumb in forward motion overcap to loosen or, using the decapping tool provided, insert toothof decapper into inner lip of cap and gently rotate the tool
backwards.

16

Agilent Seahorse XFp Mito Fuel Flex Test Kit User Manual

Assay Workflow

Figure 6

Removing reagent cap

Prepare stock compounds
Important: Use compounds the same day they are reconstituted.
Do not refreeze. Discard any remaining compound. Refer to“Removing reagent caps” on page 16 for instructions on
removing the reagent caps.

Table 3

1

Remove one foil pouch from Agilent Seahorse XFp Mito Fuel
Flex Test Kit box.

2

Remove three tubes from the pouch, and place in a small
tube rack.

3

Resuspend contents of each tube with prepared assay
medium in volumes described in Table 2 with a pipette.
Place cap on tube, and vortex for 1 minute to solubilize thecompounds.

Stock solution preparationCompound nameVolume of assay
medium (µL)

Stock concentration(µM)

10x Port
concentration (µM)

Final assay wellconcentration (µM)

BPTES

100

60

30

3.0

Etomoxir

100

80

40

4.0

UK5099

100

40

20

2.0

Prepare compounds for loading in sensor cartridge ports
Chose ONE fuel pathway to assay then make the appropriate
dilutions in a microfuge tube using Table 4.

Agilent Seahorse XFp Mito Fuel Flex Test Kit User Manual

17

Assay Workflow

Table 4Compound preparation for a dependency or flexibility test
Dilution plan
(Choose ONE)



Tube



Contents


VolumeBPTES (µL)

VolumeVolumeEtomoxir (µL) UK5099 (µL)

Volume
assay
Total volume
medium (µL) (µL)

Glutamineoxidation

1

BPTES

90

-

-

90

180

2

Etomoxir/UK5099 -

90

90

-

180

Fatty acidoxidation

1

Etomoxir

-

90

-

90

180

2

BPTES/UK5099

90

-

90

-

180

Glucoseoxidation

1

UK5099

-

-

90

90

180

2

BPTES/Etomoxir

90

90

-

-

180

Load sensor cartridge
Load the sensor cartridge for either a Dependency or Flexibility
assay using Table 5.

NOTE

For a dependency assay, only the Dependency group is needed for the fuelof interest. (This allows for the second group to be a pairwise comparison.)For a flexibility assay, both Dependency and Capacity groups are neededfor each fuel of interest.

Table 5Compound loading

Assay group

Tube

Contents

Port

VolumeGlutaminedependency

1

BPTES

A

20 µL

2

Etomoxir/UK5099

B

22 µL

Glutamine capacity

2

Etomoxir/UK5099

A

20 µL

1

BPTES

B

22 µL

Etomoxir

A

20 µL

2

BPTES/UK5099

B

22 µL

2

BPTES/UK5099

A

20 µL

1

Etomoxir

B

22 µL

1

UK5099

A

20 µL

2

BPTES/Etomoxir

B

22 µL

2

BPTES/Etomoxir

A

20 µL

1

UK5099B

22 µL

Fatty acid dependency 1

Fatty acid capacity

Glucose dependency

Glucose capacity

18

Agilent Seahorse XFp Mito Fuel Flex Test Kit User Manual

Assay Workflow

Running the Assay
1

Press Start.

2

Select the Agilent Seahorse XFp Mito Fuel Flex Test - Dependency
or Seahorse XFp Mito Fuel Flex Test - Flexibility Assay Templatefrom the Local tab.

3

Group Page: No action required - Confirm or adjust thegroups and plate map, then press the right arrow.

4

Protocol Page: No action required - Confirm the protocol,then press the right arrow.

5

Summary Page: Change the name of the assay results file, if
desired. Press Start Assay when ready.

6

Place the utility plate with the loaded assay cartridge on the
instrument tray, and click Continue. Calibration will takeapproximately 20 minutes.

7

When calibration has been completed, remove the utility
plate and place the Agilent Seahorse XFp Miniplate on thetray, and click Continue to start the assay.

Data Analysis
Of particular importance for the best quality data are items
such as optimal cell density, consistent cell seeding (as reflected
in CVs of absolute baseline rates among similarly treated wells),
and stable baseline OCR values of the cell line/type being usedin the assay. For all XF assays, significant variability between
wells within the same group indicates the need for additional
optimization of cell culture, cell seeding, orintervention/treatment conditions. If variability is high within agroup, please review the Basic Procedures for your analyzer. For
additional questions, please contact Technical Support.
After ensuring adequate data quality, proceed with data
analysis using the Seahorse XF Mito Fuel Flex Test Report
Generator. This report generator calculates the parameters of%Dependency, %Capacity, and %Flexibility with respect to eachgroup or fuel tested. These parameters provide a relative
comparison of mitochondrial fuel oxidation among groups
during basal respiration conditions. It is also encouraged thatabsolute OCR values (in pmol/min) of control and experimentalgroups are reviewed for any significant changes in OCR baseline
values between groups (measurement 3/measurement prior to
any injection). These differences in OCR values between groups
suggest some biological change, and should be incorporated into

Agilent Seahorse XFp Mito Fuel Flex Test Kit User Manual

19

Assay Workflow

the final interpretation of Mito Fuel Flex Test results.
Export to the Seahorse XF Mito Fuel Flex Test Report
Generator using Wave Desktop to generate a one-page Summary
Report. The Report Generator automatically calculates percent
Dependency, Capacity, and Flexibility, providing a simple,
standardized output for analysis and interpretation of Seahorse
XF Mito Fuel Flex Test data, and supports data analysis from all
Seahorse XF Analyzers.
To download the Seahorse XF Mito Fuel Flex Test Report
Generator and accompanying user guide visit:https://www.agilent.com/en/products/cell-analysis/cell-analysis-software/data-analysis/seahorse-xf-mito-fuel-flex-test-reportgenerator

20

Agilent Seahorse XFp Mito Fuel Flex Test Kit User Manual

Agilent Seahorse XFp Mito Fuel Flex Test Kit
User Manual4
Frequently Asked Questions

What if all three inhibitors only cause a small decrease intotal OCR?
Processes other than oxidation of these three fuels may
contribute to baseline OCR. These processes may be broken
down further into mitochondrial and nonmitochondrial oxygen
consuming processes:
Other mitochondrial respiration: respiration dependent on analternative substrate(s) being oxidized to support
mitochondrial respiration, which may include (but not limitedto) short and medium chain fatty acids and amino acids otherthan glutamine.
Nonmitochondrial oxygen consumption: consumption of oxygenby other biochemical processes in the cell. This includes (but is
not limited to) very long chain fatty acids that get partially
oxidized in the peroxisomes and other cellular enzymatic
processes that consume oxygen. The nonmitochondrial fractionof total oxygen consumption can be measured using the
Seahorse XF Cell Mito Stress Test.
Why is Dependency reported as zero?
If Dependency is not significantly above zero or negative due towell to well variability, there is no dependency on thatparticular substrate. If the cells are not dependent on the target
fuel pathway, OCR may slightly increase following injection of
inhibitor. When this occurs, Dependency is automatically set tozero (no dependence), and Flexibility will be equal to Capacity.
What does it mean if I have negative flexibility values?
When changes in OCR are small, well to well variability mightlead to negative flexibility values. Negative flexibility values ofless than 5 % are generally attributable to noise in the assay. If
you detect significant negative flexibility, contact TechnicalSupport.

Agilent Technologies

21Frequently Asked Questions

How can I further diagnose or troubleshoot Mito Fuel Flex
Test results?
The above potential issues described (apparent low response toinhibitors, 0 % dependency, or negative flexibility values) may
be further diagnosed by performing (as a separate test or addedgroup) a Mito Fuel Flex Test with only media injections (noinhibitors) to establish if baseline respiration changes
significantly over the course of the assay. If the absoluterespiration rates (OCR) in the absence of inhibitors trend
significantly upward or downward throughout the assay, thenthe parameters of this test (relative % dependency, capacity andflexibility) will be either underestimated or exaggerated,
respectively. This depends on the magnitude and direction of
any baseline trends versus the magnitude of change due toadded inhibitors. To limit any upward/downward baseline
trending OCR, ensure that cell culture and technical parametersof the assay have been thoroughly optimized.
Will these inhibitors and concentrations work with all cells?
Yes, the test uses all three compounds at concentrations well
above their EC50 values for inhibition in mammalian cells.
These values have been validated in a variety of cell lines andprimary isolates. While most cell types or cell lines have anappreciable response to at least one inhibitor, not all cells will
respond to all inhibitors. If the cells are not responsive to aparticular inhibitor, they may not be dependent on thatparticular fuel pathway (that is, they are flexible with respect tothe fuel used for oxidative phosphorylation).
How do I interpret ECAR and glycolysis in this assay?
Using combinations of inhibitors can confound interpretation of
ECAR data with this test due to shifts in cellular ATP
production and demand. For directly measuring glycolytic
function, we recommend using the Seahorse XF Glycolysis
Stress Test.
The recommended assay medium does not include fatty acid,can I add it?
Although not required, long chain fatty acid may be added tothe medium. We recommend using a single species of long chain
fatty acid, such as Seahorse XF Palmitate-BSA FAO Substrate,
when testing exogenous fatty acid oxidation. NOTE: only
oxidation of long-chain fatty acid, such as palmitate, is sensitive
to inhibition by etomoxir.

22

Agilent Seahorse XFp Mito Fuel Flex Test Kit User Manual

Agilent Technologies© Agilent Technologies, Inc.
Printed in USA, April 2019
Revision F0
For research use onlyNot for use in diagnostic procedures*103270-400*
103270-400