Agilent Seahorse XFp
Cell Mito Stress Test Kit

User Guide
Kit 103010-100

Agilent Technologies

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Manual Part Number
103011-400

Kit Part Number
103010-100

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

8

Kit Information
Kit Contents

9

Kit Shipping and Storage
Additional Required Items

910Assay
Day Prior to Assay

12

Day of the Assay

12

Run the Assay

18

Data Analysis

18

Agilent Seahorse XFp Cell Mito Test Kit User Guide

34Agilent Seahorse XFp Cell Mito Test Kit User Guide

Agilent Seahorse XFp Cell Mito Stress Test Kit
User Guide

1Introduction
Assay Background 5
Glossary 8

Assay Background
The Agilent Seahorse XFp Cell Mito Stress Test measures key
parameters of mitochondrial function by directly measuring theoxygen consumption rate (OCR) of cells on the Seahorse XFp
Extracellular Flux Analyzers. It is a plate-based live cell assaythat enables monitoring OCR in real time.
The assay uses the built-in injection ports on XFp sensor
cartridges to add modulators of respiration into cell wells
during the assay to reveal the key parameters of mitochondrial
function. The modulators included in this assay kit are
Oligomycin, Carbonyl cyanide-4 (trifluoromethoxy)
phenylhydrazone (FCCP), Rotenone, and Antimycin. Figure 1
illustrates the injection sequence of these modulators and theparameters can be obtained with this assay. More detailed
description or definition of these parameters is provided in the“Glossary” on page 8 and the “Reference” on page 7 by
Divakaruni et al, 2014.

Figure 1

Seahorse XFp Cell Mito Stress Test profile, showing the key
parameters of mitochondrial function.

Agilent Technologies

5Introduction

Figure 2 illustrates the complexes of the Electron Transport
Chain (ETC), and indicates the target of action for all themodulators included in the Seahorse XFp Cell Mito Stress TestKit.

Figure 2Agilent Seahorse XFp Cell Mito Stress Test modulators of theETC

Oligomycin inhibits ATP synthase (complex V), and is injected
first in the assay following basal measurements. It impacts ordecreases electron flow through the ETC, resulting a reduction
in mitochondrial respiration or OCR. This decrease in OCR islinked to cellular ATP production.
Carbonyl cyanide-4 (trifluoromethoxy) phenylhydrazone(FCCP) is an uncoupling agent that collapses the proton
gradient and disrupts the mitochondrial membrane potential. It
is the 2nd injection following Oligomycin. As a result, electron
flow through the ETC is uninhibited, and oxygen consumption
by complex IV reaches the maximum. The FCCP-stimulated OCR
can then be used to calculate spare respiratory capacity,
defined as the difference between maximal respiration andbasal respiration. Spare respiratory capacity is a measure of theability of the cell to respond to increased energy demand or
under stress.
The third injection is a mixture of rotenone, a complex I
inhibitor, and antimycin A, a complex III inhibitor. This
combination shuts down mitochondrial respiration and enables
the calculation of nonmitochondrial respiration driven byprocesses outside the mitochondria.
Table 1 provides a summary of these effects.

6

Agilent Seahorse XFp Cell Mito Test Kit User GuideIntroduction

Table 1

Summary of target and effect for the mitochondrial respiration
modulatorsCompound(s)ETC target

Effect on OCR

Oligomycin

ATP synthase (complex V)

Decrease

FCCP

Inner mitochondrial membrane

Increase

Rotenone/antimycin A

Complex I and III (respectively)

Decrease

The ability to assess mitochondrial function has enabledresearchers to advance their understanding of metabolism’s keyrole in cellular physiology, disease pathology, and etiology. TheSeahorse XFp Cell Mito Stress Test is the gold standard assay,
and is widely used for measuring mitochondrial function incells. This assay provides insight into the cause ofmitochondrial dysfunction and an in-depth understanding of
metabolic pathways, signals, and phenotypes.

Reference
Divakaruni AS, Paradyse A, Ferrick DA, Murphy AN,
Jastroch M. 2014. Analysis and Interpretation of
Microplate-Based Oxygen Consumption and pH data. In
Methods in Enzymology, Volume 547, Chapter 16, 309-354.

Agilent Seahorse XFp Cell Mito Test Kit User Guide7

Introduction

Glossary• Basal respiration: Oxygen consumption used to meetcellular ATP demand resulting from mitochondrial protonleak. Shows energetic demand of the cell under baseline
conditions.
• ATP Production: The decrease in oxygen consumption rate
upon injection of the ATP synthase inhibitor oligomycin
represents the portion of basal respiration that was being
used to drive ATP production. Shows ATP produced by themitochondria that contributes to meeting the energetic
needs of the cell.
• H+ (Proton) leak: Remaining basal respiration not coupled
to ATP production. Proton leak can be a sign ofmitochondrial damage, or can be used as a mechanism toregulate the mitochondrial ATP production.
• Maximal respiration: The maximal oxygen consumption
rate attained by adding the uncoupler FCCP. FCCP mimics aphysiological “energy demand” by stimulating therespiratory chain to operate at maximum capacity, whichcauses rapid oxidation of substrates (sugars, fats, and aminoacids) to meet this metabolic challenge. Shows themaximum rate of respiration that the cell can achieve.
• Spare respiratory capacity: This measurement indicatesthe capability of the cell to respond to an energetic demand
as well as how closely the cell is to respiring to its
theoretical maximum. The cell's ability to respond todemand can be an indicator of cell fitness or flexibility.
• Nonmitochondrial respiration: Oxygen consumption thatpersists due to a subset of cellular enzymes that continue toconsume oxygen after the addition of rotenone andantimycin A. This is important to get an accurate measure ofmitochondrial respiration.

8Agilent Seahorse XFp Cell Mito Test Kit User Guide

Agilent Seahorse XFp Cell Mito Stress Test Kit
User Guide

2Kit Information
Kit Contents 9
Kit Shipping and Storage 9
Additional Required Items 10

Kit Contents
The Seahorse XFp Cell Mito Stress Test Kit includes six foil
pouches that each contain reagents sufficient for a complete
Seahorse XFp Cell Mito Stress Test in the Agilent Seahorse XFp
Cell Culture Miniplate.
Every pouch includes one tube of each of the following
compounds: oligomycin, FCCP and a mix ofrotenone/antimycin A as indicated in Table 2.
Table 2

Agilent Seahorse XFp Cell Mito Stress Test Kit foil pouchcontentsCompoundCap color

Quantity per tube(nmol)

Oligomycin

Blue

12.6

FCCP

Yellow

14.4

Rotenone + antimycin ARed

5.4 (of both)

Kit Shipping and Storage
The product ships at ambient temperature. The product can bestored at room temperature, and is stable for one year from thedate of manufacture. The expiration date is printed on the labelof the kit box. Depending on the shipping date, the actual shelf
life of the kit in the user’s hand can vary between
12 to 3 months.

Agilent Technologies

9Kit Information

Additional Required Items
The following items are also required for performing Seahorse
XFp Mito Stress Tests, but they are not supplied with the kits.
Table 3

Additional required items

Items

Supplier

Catalog number

Agilent Seahorse XFp Analyzers

Agilent TechnologiesSeahorse XFp FluxPak (cartridges,miniplates, and calibrant)

Agilent Technologies 103022-100

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

103575-100
Agilent Technologies 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* 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 theSeahorse XF Media Selection Guide.
http://www.agilent.com/cs/library/selectionguide/public/5991-7878EN.pdf

10Agilent Seahorse XFp Cell Mito Test Kit User Guide

Agilent Seahorse XFp Cell Mito Stress Test Kit
User Guide

3AssayDay Prior to Assay 12
Day of the Assay 12
Run the Assay 18
Data Analysis 18

Figure 3

NOTE

Agilent Seahorse XFp Cell Mito Stress Test Assay Workflow

Optimal cell seeding density and FCCP concentration should be empiricallydetermined for your cell type prior to the assay. For more details, pleaserefer to the Basic Procedures on Agilent Cell Analysis Learning Center.
www.agilent.com/en/products/cell-analysis/how-to-run-an-assay
The Cell Line Reference Database is a good resource for finding
information regarding the cell type or interest.
www.agilent.com/cell-reference-database

Agilent Technologies

11AssayDay Prior to Assay
1

Turn on the Agilent Seahorse XFp Analyzer, and let it warm
up overnight (minimum five hours).

2

Plate the cells at a previously determined optimized density
in the Seahorse XFp Cell Culture Miniplate using theappropriate cell culture growth medium.

3Hydrate a sensor cartridge in Agilent Seahorse XF Calibrantat 37 °C in a non-CO2 incubator overnight.

Day of the Assay
Prepare assay medium

121

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.

Agilent Seahorse XFp Cell Mito Test Kit User GuideAssay

Prepare compound stock solutions and working solutions
Important: Use the compounds on the same day that they arereconstituted. Discard any remaining compound solutions. Do
not refreeze and reuse. Materials in each pouch are sufficientfor one miniplate assay.
1

Remove one foil pouch and the decapper from Seahorse XFp
Cell Mito Stress Test Kit box.

2

Open the pouch, and remove the three tubes containing
oligomycin (blue cap), FCCP (yellow cap), androtenone/antimycin A (red cap) with glove hand. Place thetubes in a small tube rack.

3

Remove the cap of each tube by inserting the tooth of thedecapper into the inner lip of the cap, and gently rotate thetool backwards. See Figure 4.

Figure 4

Removing reagent caps

4

Resuspend content in each tube with prepared assay
medium in volumes described in Table 4.

5

Using a pipette, gently pipette up and down the medium(~10 times) to solubilize the compounds. These are thecompound stock solutions.

Table 4

Stock solutions.

CompoundVolume of assay medium

Stock concentration

Oligomycin

280 µL

45 µM

FCCP

288 µL

50 µM

Rot/AA

216 µL

25 µM

6Use the compound stock solutions to make compound
working solutions for loading into the injection ports onsensor cartridges.

Agilent Seahorse XFp Cell Mito Test Kit User Guide

13

AssayIt is recommended to use the constant compoundconcentration with variable loading volume approach. Thisapproach entails preparing the compound working solutions
at a constant concentration, and requires that a different
volume for each compound is loaded in the injection port.
7

Prepare 0.3 mL working solutions for each compound inassay medium, using the volumes indicated in Table 5 for
XFp analyzers.
The optimal final compound concentration for achieving
maximal effect is cell line dependent, and may be affected byassay medium types. Therefore, it is recommended that, foreach new cell line or assay medium, a titration experiment
for the compounds is performed. This is especially
important with FCCP, as the titration curve tends to be quite
sharp, and too much FCCP can actually diminish responses
in OCR. Follow the concentration range provided in Table 5
to set up the experiment. For Oligomycin, 1.5 µM isrecommended for most cell types, while for Rot/AA, 0.5 µM
is recommended. For questions, please contact Agilent CellAnalysis Technical Support.

Table 5Compound preparation for loading to XFp sensor cartridges.
Starting assay medium volume for cell plate is 180 µL per well.

Port
Final well
designation (µM)

StockMediasolution
volumevolume (µL) (µL)

10X Port(µM)

Volumeadded toport (µL)

Port A
Oligomycin

1.51002001520Port BFCCP0.125

7.5

292.5

1.25

220.25

15285

2.5

220.5

30

270

5

221.060240

10222.0120

180

20

220.5

60

240

525Port C
Rot/AALoad solutions into the ports on sensor cartridge
Proper port-loading techniques can be found in Basic
Procedure, “Loading the Sensor Cartridge with Compounds”, onthe Agilent Cell Analysis Learning Center.
www.agilent.com/en/products/cell-analysis/how-to-run-an-assay
Please read the information prior to loading compounds.
14Agilent Seahorse XFp Cell Mito Test Kit User GuideAssayEnsure that the sensor cartridge is properly hydrated prior touse.
For location of the ports, please refer to Figure 5.

Figure 5

Location and numbering of injection ports on sensor cartridges

There are two types of assays that can be performed:• Standard Assay - Only involves the injection of modulators
included in the kit.
• Modified Assay - Includes an additional injection of a test
compound prior to oligomycin injection, and Port A is used
for the testing compound.
Refer to Table 6 for loading volume and port designation forcompounds in different types of assays.
Table 6Port

Assay types, injection Port, and volume recommendation.
Starting assay medium volume for cell plate is 180 µL per well.
StandardassayModifiedassay

Port
Add to port volumeconcentration (µL)

A

Oligomycin Test
compound*

10X

20

B

FCCP

Oligomycin

10X

22

C

Rot/AA

FCCP

10X

25

D—Rot/AA

10X

27* For negative controls, assay medium should be used to replace the test compound.

Prepare Seahorse XFp Cell Culture Miniplate for assay
1Remove cell culture miniplates from a 37 °C CO2 incubator,and examine the cells under a microscope to confirm
confluence.

2

Remove the assay medium from water bath.

3

Change the cell culture growth medium in the cell culture
miniplate to warmed assay medium using a multichannel
pipette, and place the cell culture miniplate into a 37 °C
non-CO2 incubator for 45 minutes to 1 hour prior to theassay.

Agilent Seahorse XFp Cell Mito Test Kit User Guide

15

AssayRun the Assay
Run the Agilent Seahorse XFp Cell Mito Stress Test

NOTE

1

Click Start, and select the Agilent Seahorse Cell Mito Stress Testdefault template. Click the right arrow on the Groups page,and then on the Protocol page, click Start Assay.

2Place the utility plate with the loaded assay cartridge on theinstrument tray, and click Continue. Calibration takes
approximately 20 minutes.

Remove the cartridge lid, and verify correct plate orientation.

3

NOTE

When calibration has been completed, remove the utility
plate and place the Seahorse XFp Cell Culture Miniplate on
the tray, and press continue to start the assay.

Remove the miniplate lid, and verify correct plate orientation.

Data Analysis
The Agilent Seahorse XF Cell Mito Stress Test Report Generator
automatically calculates the Agilent Seahorse XFp Cell Mito
Stress Test parameters from Wave data that has been exported
to Excel. The Seahorse XF Stress Test Report Generator can beused with either a standard or modified stress test protocol,
and provides a convenient, customizable, one-page assay
summary.
The Report Generator can be installed either alongside Wave or
directly from the Agilent Cell Analysis website. Visit
https://www.agilent.com/en/products/cell-analysis/cell-analysis-software/data-analysis/seahorse-xf-cell-mito-stress-test-reportgenerators to learn more about the Seahorse XF Stress Test
Report Generators, and download the User Guide.

16Agilent Seahorse XFp Cell Mito Test Kit User Guide

Agilent Technologies© Agilent Technologies, Inc.
Printed in USA, April 2019
Revision F0
For research use onlyNot for use in diagnostic procedures*103011-400*
103011-400