Agilent Seahorse XFp

Mito Fuel Flex Test Kit





For use with Agilent Seahorse
XFp Extracellular Flux Analyzers

User Manual 
Kit 103270-100

Agilent Technologies
Notices
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103270-400
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103270-100
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Contents
Introduction
Assay Background 5
Glossary 10

Kit Information
Kit Contents 11
Kit Shipping and Storage 11
Additional Required Items 12

Assay Workflow
Day Prior to Assay 14
Day of Assay 14
Running the Assay 18
Data Analysis 19

Frequently Asked Questions

Agilent Seahorse XFp Mito Fuel Flex Test Kit User Manual 3
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 for
measuring 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 of
oxidation of each fuel by measuring mitochondrial respiration
[the oxygen consumption rate, OCR] of cells in the presence or
absence of fuel pathway inhibitors (Figure 1 on page 6).
Sequentially inhibiting the pathway of interest followed by the
two 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 that
the cells’ mitochondria are unable to compensate for the
blocked pathway by oxidizing other fuels. Inhibiting the two
alternative pathways followed by the pathway of interest
enables the calculation of cells' mitochondrial capacity to meet
energy 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 to
compensate for the inhibited pathway by utilizing other
pathways to fuel mitochondrial respiration. The presence of
dependency 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, and
flexibility 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 and
oxidized 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 is
then 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 beta
oxidation.

Figure 1 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 these
fuel sources is determined by measuring the decrease in fuel
oxidation (decline in oxygen consumption rate) upon addition
of one or more inhibitors.

6 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 the
target 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 injected
following the third rate measurement (“Baseline”
measurement). The sixth rate measurement following this
injection is used for the calculation to allow sufficient time for
the compound to have a complete effect (“Target Inhibitor”
measurement). The final rate measurement (“All Inhibitors”) is
used to complete the equation.

Agilent Seahorse XFp Mito Fuel Flex Test Kit User Manual 7
Introduction

Figure 3 Fuel Capacity: Glutamine Oxidation Pathway Example

Fuel Capacity is tested by first injecting inhibitors of the
alternative 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 injected
following the third rate measurement (“Baseline”
measurement). The sixth rate measurement following this
injection is used for the calculation to allow sufficient time for
the compounds to have a complete effect (“Other 2 Inhibitors”
measurement). The final rate measurement (“All Inhibitors”) is
used to complete the equation.

8 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 the
difference between Capacity and Dependency. All three
parameters (Dependency, Capacity, and Flexibility) are
displayed as a stacked bar chart when using the Agilent
Seahorse XF Mito Fuel Flex Test Report Generator. See “Data
Analysis” on page 19 for further information.

Agilent Seahorse XFp Mito Fuel Flex Test Kit User Manual 9
Introduction

Glossary

Baseline Respiration Rate of oxygen consumption due to fuel

oxidation under initial assay conditions.

Fuel A substrate or nutrient that is used by cells and oxidized

in the mitochondria. In this assay, the mitochondrial oxidation
of 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 transport
of pyruvate into mitochondria).

Fuel Dependency The measurement of cells' reliance on a

particular fuel pathway to maintain baseline respiration.

Fuel Capacity The ability of a cell's mitochondria to oxidize a

fuel when other fuel pathways are inhibited.

Fuel Flexibility The difference between fuel capacity and

dependency, that is, the ability of cells to increase oxidation of a
particular fuel in order to compensate for inhibition of
alternative fuel pathway(s).

10 Agilent Seahorse XFp Mito Fuel Flex Test Kit User Manual
 Agilent Seahorse XFp Mito Fuel Flex Test Kit
User Manual

2
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 on
page 13). The kit includes six foil pouches, each pouch contains
one tube of each of the following compounds.

Table 1 Kit compounds

Compound Cap color Amount per tube (nmol)

BPTES Grey 6
Etomoxir Green 8
UK5099 Orange 4

Kit Shipping and Storage

Product ships at ambient temperature in an insulated cooler
box. Product should be stored at -20 °C for up to one year from
the date of manufacture (listed on package).

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 Agilent
XFp Analyzer Technologies
Agilent Seahorse Agilent 103193-100
XF Base Medium, Technologies
100 mL
Agilent Seahorse Agilent 103022-100
XFp FluxPak Technologies
100 mM Pyruvate Sigma S8636 or
equivalent
200 mM Glutamine Sigma G8540 or
equivalent
2.5 M Glucose Sigma G8769 or
equivalent
Microfuge tubes Various 0.5-1.5 mL capacity

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 18
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 the
appropriate 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:
http://www.agilent.com/en-us/products/cell-analysis-(seaho
rse)/basic-procedures-to-run-an-xf-assay
2 Hydrate a sensor cartridge in Agilent Seahorse XF Calibrant
at 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 from
the 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.

http://www.seahorsebio.com/support/software/report-gene
rators.php
The Dependency or Flexibility assay may also be set up
directly on the Agilent Seahorse XFp Analyzer. First, select
the 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
1 Turn on the Agilent Seahorse XFp Analyzer and let it warm
up (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 a
pairwise comparison of fuel dependency, or a single cell
condition with both dependency and flexibility.

14 Agilent Seahorse XFp Mito Fuel Flex Test Kit User Manual
 Assay Workflow

Prepare Assay medium

1 Prepare assay medium by supplementing Agilent Seahorse
XF Base Medium. Seahorse recommends 1 mM pyruvate,
2 mM glutamine, and 10 mM glucose as a starting point;
however, desired medium composition can be varied
depending on cell type or in vitro culture conditions. Refer
to Basic Procedures for the Agilent Seahorse XFp Analyzer.
2 Warm assay medium to 37 °C.
3 Adjust pH to 7.4 with 0.1 N NaOH (Note: sterile filtration
following pH adjustment is recommended).
4 Keep assay medium at 37 °C until ready to use.

Prepare Agilent Seahorse XFp Cell Culture Miniplate for Assay

• Adherent cells
1 Remove cell culture miniplate from 37 °C CO2 incubator
and examine cells under microscope to confirm that cells
are 50-90 % confluent and evenly distributed within each
well.
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 volume
of 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 at
the 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.
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.

Agilent Seahorse XFp Mito Fuel Flex Test Kit User Manual 15
Assay Workflow

Load template onto the Agilent Seahorse XFp Analyzer

(If template(s) already present, skip this step.)
1 Visit the Seahorse Bioscience website, and download the
Agilent Seahorse XF Mito Fuel Flex Report Generator along
with both Dependency and Flexibility Assay Templates.
Both Seahorse XFp Mito Fuel Flex Test - Dependency and
Seahorse 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 be
imported, then click Import.
7 All imported Assay Templates will now be available for
selection from the Local tab when starting an XFp assay.

Removing reagent caps

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

Figure 6 Removing reagent cap

16 Agilent Seahorse XFp Mito Fuel Flex Test Kit User Manual
 Assay Workflow

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.
1 Remove one foil pouch from Agilent Seahorse XFp Mito Fuel
Flex Test Kit box. Return box with remaining kits to -20 °C.
2 Remove three tubes from the pouch, and place in a small
tube rack. Allow compounds to warm to room temp for
approximately 15 minutes.
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 the
compounds.
Table 3 Stock solution preparation

 Volume of assay Stock concentration 10x Port Final assay well

Compound name medium (µL) (µM) concentration (µM) concentration (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.
Table 4 Compound preparation for a dependency or flexibility test

      Volume 
Dilution plan   Volume Volume Volume assay Total volume
(Choose ONE) Tube Contents BPTES (µL) Etomoxir (µL) UK5099 (µL) medium (µL) (µL)
Glutamine 1 BPTES 90 - - 90 180
oxidation
2 Etomoxir/UK5099 - 90 90 - 180
Fatty acid 1 Etomoxir - 90 - 90 180
oxidation
2 BPTES/UK5099 90 - 90 - 180
Glucose 1 UK5099 - - 90 90 180
oxidation
2 BPTES/Etomoxir 90 90 - - 180

Agilent Seahorse XFp Mito Fuel Flex Test Kit User Manual 17
Assay Workflow

Load sensor cartridge

Load the sensor cartridge for either a Dependency or Flexibility
assay using Table 5.
For a dependency assay, only the Dependency group is needed for the fuel
NOTE
of interest. (This allows for the second group to be a pairwise comparison.)
For a flexibility assay, both Dependency and Capacity groups are needed
for each fuel of interest.

Table 5 Compound loading

Assay group Tube Contents Port Volume

Glutamine 1 BPTES A 20 µL
dependency
2 Etomoxir/UK5099 B 22 µL
Glutamine capacity 2 Etomoxir/UK5099 A 20 µL
1 BPTES B 22 µL
Fatty acid dependency 1 Etomoxir A 20 µL
2 BPTES/UK5099 B 22 µL
Fatty acid capacity 2 BPTES/UK5099 A 20 µL
1 Etomoxir B 22 µL
Glucose dependency 1 UK5099 A 20 µL
2 BPTES/Etomoxir B 22 µL
Glucose capacity 2 BPTES/Etomoxir A 20 µL
1 UK5099 B 22 µL

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 Template
from the Local tab.
3 Group Page: No action required - Confirm or adjust the
groups 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.

18 Agilent Seahorse XFp Mito Fuel Flex Test Kit User Manual
 Assay Workflow

6 Place the utility plate with the loaded assay cartridge on the
instrument tray, and click Continue. Calibration will take
approximately 20 minutes.
7 When calibration has been completed, remove the utility
plate and place the Agilent Seahorse XFp Miniplate on the
tray, 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 used
in 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, or
intervention/treatment conditions. If variability is high within a
group, 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 each
group or fuel tested. These parameters provide a relative
comparison of mitochondrial fuel oxidation among groups
during basal respiration conditions. It is also encouraged that
absolute OCR values (in pmol/min) of control and experimental
groups 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
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:
http://www.agilent.com/en-us/products/cell-analysis-(seahorse
)/report-generator-for-the-xf-mito-fuel-flex-test

Agilent Seahorse XFp Mito Fuel Flex Test Kit User Manual 19
Assay Workflow

20 Agilent Seahorse XFp Mito Fuel Flex Test Kit User Manual
Agilent Seahorse XFp Mito Fuel Flex Test Kit
User Manual

4
Frequently Asked Questions

What if all three inhibitors only cause a small decrease in

total 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 an
alternative substrate(s) being oxidized to support
mitochondrial respiration, which may include (but not limited
to) short and medium chain fatty acids and amino acids other
than glutamine.
Nonmitochondrial oxygen consumption: consumption of oxygen
by 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 fraction
of 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 to
well to well variability, there is no dependency on that
particular 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 to
zero (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 might
lead to negative flexibility values. Negative flexibility values of
less than 5 % are generally attributable to noise in the assay. If
you detect significant negative flexibility, contact Technical
Support.

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 to
inhibitors, 0 % dependency, or negative flexibility values) may
be further diagnosed by performing (as a separate test or added
group) a Mito Fuel Flex Test with only media injections (no
inhibitors) to establish if baseline respiration changes
significantly over the course of the assay. If the absolute
respiration rates (OCR) in the absence of inhibitors trend
significantly upward or downward throughout the assay, then
the parameters of this test (relative % dependency, capacity and
flexibility) will be either underestimated or exaggerated,
respectively. This depends on the magnitude and direction of
any baseline trends versus the magnitude of change due to
added inhibitors. To limit any upward/downward baseline
trending OCR, ensure that cell culture and technical parameters
of 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 and
primary isolates. While most cell types or cell lines have an
appreciable response to at least one inhibitor, not all cells will
respond to all inhibitors. If the cells are not responsive to a
particular inhibitor, they may not be dependent on that
particular fuel pathway (that is, they are flexible with respect to
the 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 to
the 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, March 2017
Revision C0
For research use only
Not for use in diagnostic procedures

*103270-400*
103270-400