Seahorse XF Cell Mito Stress Kit User Guide
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Agilent Technologies
Agilent Seahorse XF Cell
Mito Stress Test Kit
User Guide
Kit 103015-100
Notices
© Agilent Technologies, Inc. 2017
No part of this manual may be reproduced
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tion into a foreign language) without prior
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United States and international copyright
laws.
Manual Part Number
103016-400
Kit Part Number
103015-100
Edition
First edition, March 2017
Revision E0
Printed in USA
Agilent Technologies, Inc.
2850 Centerville Road
Wilmington, DE 19808-1610 USA
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4 Agilent Seahorse XF Cell Mito Stress Test Kit User Guide
5
Agilent Seahorse XF Cell Mito Stress Test Kit
User Guide
Agilent Technologies
1
Introduction
Assay Background 5
Glossary 8
Assay Background
The Agilent Seahorse XF Cell Mito Stress Test measures key
parameters of mitochondrial function by directly measuring the
oxygen consumption rate (OCR) of cells. Sequential compound
injections measure basal respiration, ATP production, proton
leak, maximal respiration, spare respiratory capacity, and
nonmitochondrial respiration (see Figure 1 on page 6). The
Seahorse XF Cell Mito Stress Test uses modulators of
respiration that target components of the electron transport
chain (ETC) in the mitochondria to reveal key parameters of
metabolic function. The compounds (oligomycin, FCCP, and a
mix of rotenone and antimycin A) are serially injected to
measure ATP production, maximal respiration, and
nonmitochondrial respiration, respectively. Proton leak and
spare respiratory capacity are then calculated using these
parameters and basal respiration.
Figure 2 on page 6 illustrates the complexes of the ETC and the
target of action of all of the compounds in the Seahorse XF Cell
Mito Stress Test Kit. Oligomycin inhibits ATP synthase
(complex V), FCCP uncouples oxygen consumption from ATP
production, and rotenone and antimycin A inhibit complexes I
and III, respectively.
Table 1 on page 7, describes how each modulator targets a specific
component of the ETC. Oligomycin inhibits ATP synthase
(complex V), and the decrease in OCR following injection of
oligomycin correlates to the mitochondrial respiration associated
with cellular ATP production. Carbonyl cyanide-4
(trifluoromethoxy) phenylhydrazone (FCCP) is an uncoupling
agent that collapses the proton gradient and disrupts the
mitochondrial membrane potential. As a result, electron flow
through the ETC is uninhibited, and oxygen is maximally
consumed by complex IV.
6 Agilent Seahorse XF Cell Mito Stress Test Kit User Guide
Introduction
The FCCP-stimulated OCR can then be used to calculate spare
respiratory capacity, defined as the difference between maximal
respiration and basal respiration. Spare respiratory capacity is
a measure of the ability of the cell to respond to increased
energy demand. The third injection is a mix 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
by processes outside the mitochondria.
Figure 1 Agilent Seahorse XF Cell Mito Stress Test profile of the key
parameters of mitochondrial respiration
Figure 2 Agilent Seahorse XF Cell Mito Stress Test modulators of the
ETC
Introduction
Agilent Seahorse XF Cell Mito Stress Test Kit User Guide 7
The Seahorse XF Cell Mito Stress Test is the standard assay for
measuring mitochondrial function in cells. The Seahorse XF
Cell Mito Stress Test Kit is designed to evaluate mitochondrial
function using the Seahorse XFe and Seahorse XF Extracellular
Flux Analyzers. The kit contains the reagents required to
determine key parameters of mitochondrial function (See the
“Glossary” on page 8 for more details.)
The ability to assess mitochondrial function has enabled
researchers to advance their understanding of metabolism’s key
role in cellular physiology, disease pathology, and etiology. This
assay provides insight into the cause of mitochondrial
dysfunction and an in-depth understanding of metabolic
pathways, signals, and phenotypes.
Tab le 1 Agilent Seahorse XF Cell Mito Stress Test Reagents (in order
of injection
Compound(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
8 Agilent Seahorse XF Cell Mito Stress Test Kit User Guide
Introduction
Glossary
• Basal respiration: Oxygen consumption used to meet
cellular ATP demand and resulting from mitochondrial
proton leak. 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 the
mitochondria 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 of
mitochondrial damage or can be used as a mechanism to
regulate the mitochondrial ATP production.
• Maximal respiration: The maximal oxygen consumption
rate attained by adding the uncoupler FCCP. FCCP mimics a
physiological “energy demand” by stimulating the
respiratory chain to operate at maximum capacity, which
causes rapid oxidation of substrates (sugars, fats, amino
acids) to meet this metabolic challenge. Shows the
maximum rate of respiration that the cell can achieve.
• Spare respiratory capacity: This measurement indicates
the 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 to
demand can be an indicator of cell fitness or flexibility.
• Nonmitochondrial respiration: Oxygen consumption that
persists due to a subset of cellular enzymes that continue to
consume oxygen after rotenone and antimycin A addition.
This is important for getting an accurate measure of
mitochondrial respiration.
9
Agilent Seahorse XF Cell Mito Stress Test Kit
User Guide
Agilent Technologies
2
Kit Information
Kit Contents 9
Kit Shipping and Storage 9
Additional Required Items 10
Kit Contents
The Seahorse XF Cell Mito Stress Test Kit includes six foil
pouches that each contain reagents sufficient for a complete
Seahorse XF Cell Mito Stress Test in either the 96 or 24 well
Agilent Seahorse XF Cell Culture Microplate. Every pouch
includes one tube of each of the following compounds:
oligomycin, FCCP, and a mix of rotenone/antimycin A. See
Table 2.
Kit Shipping and Storage
Product ships at ambient temperature. Product can be stored at
room temperature and is stable for 1 year from the date of
manufacture (listed on the box).
Tab le 2 Agilent Seahorse XF Cell Mito Stress Test Kit foil pouch
contents
Compound Cap color Quantity per tube (nmol)
Oligomycin*
* Oligomycin is a mixture of Oligomycin A, B, and C with Oligomycin A~60%.
Blue 63
FCCP Yellow 72
Rotenone + antimycin A Red 27 (of both)
10 Agilent Seahorse XF Cell Mito Stress Test Kit User Guide
Kit Information
Additional Required Items
The following items are also required for performing Seahorse
XF Mito Stress Tests, but they are not supplied with the kits.
Narrow p1000 pipette tips are recommended for reconstituting
compounds within the tube provided (for example,
Fisherbrand™ SureOne™ Micropoint Pipet Tips,
p/n 02-707-402).
Item Supplier Catalog Number
Agilent Seahorse XFe/XF
Analyzers
Agilent Technologies
XF Base Medium Agilent Technologies 102353-100*
* Please refer to the Seahorse XF Media Selection Guide for a full listing of all media types
and our validated recommendations for each kit type.
http://www.agilent.com/cs/library/selectionguide/public/5991-7878EN.pdf
100 mM Pyruvate Sigma S8636 or equivalent
200 mM Glutamine Sigma G8540 or equivalent
2.5 M Glucose Sigma G8769 or equivalent
12 Agilent Seahorse XF Cell Mito Stress Test Kit User Guide
Assay Workflow
NOTE
Optimal concentrations of FCCP and cell seeding density should be
empirically determined for your cells prior to the assay. Refer to FCCP
Optimization with the Seahorse XF Cell Mito Stress Test, and Seeding Cells
in Seahorse XF Cell Culture Microplates Basic Procedures available on the
Agilent website for more information on optimization.
The Cell Line Reference Database
(www.agilent.com/cell-reference-database) is a good resource for finding
reference publications for your cell type or interest.
If you are optimizing concentrations for your assay, start with the highest
concentrations given in Ta ble 3 on page 15, and make serial dilutions to
cover the desired concentration range.
Assay Workflow
Agilent Seahorse XF Cell Mito Stress Test Kit User Guide 13
Day Prior to Assay
1Turn on the Agilent Seahorse XFe/XF Analyzer, and let it
warm up overnight (minimum of five hours).
2Plate cells at a previously determined optimized density in
the Seahorse XF Cell Culture Microplate using the
appropriate cell culture growth medium. Refer to Basic
Procedure, “Seeding Cells in Seahorse XF Cell Culture
Microplates”, available on the Seahorse Bioscience website
for more information.
www.agilent.com/en-us/products/cell-analysis-(seahorse)/b
asic-procedures-to-run-an-xf-assay
3Hydrate a sensor cartridge in Seahorse XF Calibrant at
37 °C in a non-CO2 incubator overnight. Refer to Basic
Procedure, “Hydrating the Sensor Cartridge”, on the
Seahorse website for more details.
www.agilent.com/en-us/products/cell-analysis-(seahorse)/b
asic-procedures-to-run-an-xf-assay
4Design experiment in Wave. See the Instrument User
Manual for additional details.
14 Agilent Seahorse XF Cell Mito Stress Test Kit User Guide
Assay Workflow
Day of Assay
Prepare assay medium
1Prepare assay medium by supplementing Seahorse XF Base
Medium. It is best to use 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 Procedure,
“Preparing Assay Media for Use in XF Assays”, on the
Seahorse Bioscience website for more information.
www.agilent.com/en-us/products/cell-analysis-(seahorse)/b
asic-procedures-to-run-an-xf-assay
2Warm the assay medium to 37 °C.
3Adjust the pH to 7.4 with 0.1 N NaOH.
Agilent Seahorse recommends sterile filtration following pH
adjustment.
4Keep at 37 °C until ready to use.
Removing reagent caps
1Hold the tube in gloved hand.
2Roll thumb in forward motion over the cap to loosen or,
using the decapping tool provided, insert the tooth of a
decapper into the inner lip of the cap, and gently rotate the
tool backwards. See Figure 4 on page 14.
Figure 4 Removing reagent caps.
Assay Workflow
Agilent Seahorse XF Cell Mito Stress Test Kit User Guide 15
Prepare stock compounds
Important: Use compounds the same day they are
reconstituted. Do not refreeze. Discard any remaining
compound.
The Seahorse XF Cell Mito Stress Test Kit includes six foil
pouches each containing three tubes of:
•Oligomycin (blue cap)
•FCCP (yellow cap)
•Rotenone/antimycin A (red cap)
Kit reagents are sufficient for six complete Seahorse XF Cell
Mito Stress Test assays in the Seahorse XF cell culture
microplate.
See “Removing reagent caps” on page 14 for instructions on
using the decapping tool.
1Remove a foil pouch, and allow the compounds to warm to
room temperature in the sealed pouch for approximately
15 minutes.
2Open a foil pouch, and remove the three tubes containing
oligomycin (blue cap), FCCP (yellow cap), and
rotenone/antimycin A (red cap).
3Place the tubes in a small tube rake.
4Using a pipette, resuspend the contexts of each tube with
the prepared assay medium in volumes described in Table 3.
5Gently pipette up and down (~10 times) to solubilize the
compounds.
Tab le 3 Stock solution
Compound Volume of assay medium Final stock concentration
Oligomycin 630 µL 100 µM
FCCP 720 µL 100 µM
ROT/AA 540 µL 50 µM
16 Agilent Seahorse XF Cell Mito Stress Test Kit User Guide
Assay Workflow
Prepare compounds for loading in sensor cartridge
Seahorse recommends using the constant compound
concentration with variable loading volume approach. This
approach entails preparing the compounds at a constant
concentration and requires that a different volume of each
compound be loaded in the injection port. Table 4 on page 16
and Table 5 on page 17 describe how to prepare to load the
cartridges using this method for Seahorse XFe/XF 96 analyzers
and Seahorse XFe/XF 24 analyzers, respectively.
Prepare 3 mL each compound in assay medium. Seahorse
recommends using 1 µM of oligomycin for most cells. Please
contact Technical Support with any questions.
Tab le 4 Compound Preparation for Loading Sensor Cartridge Ports for
Seahorse XFe/XF 96 analyzers. Starting with well volume of
180 µL assay medium
(Final well)
(µM)
Stock
volume
(µL)
Media
volume
(µL)
10X (Port)
(µM)
Add to
port (µL)
Port A
Oligomycin
0.5 150 2,850 5 20
1.0 300 2,700 10 20
2.0 600 2,400 20 20
Port B
FCCP
0.125 37.5 2,962.5 1.25 22
0.25 75 2,925 2.5 22
0.5 150 2,850 5 22
1.0 300 2,700 10 22
2.0 600 2,400 20 22
Port C
Rotenone/
antimycin A
0.5 300 2,700 5 25
Assay Workflow
Agilent Seahorse XF Cell Mito Stress Test Kit User Guide 17
Load sensor cartridge
Refer to Basic Procedure, “Loading the Sensor Cartridge with
Compounds”, on the Seahorse Bioscience website for proper
port-loading technique.
www.agilent.com/en-us/products/cell-analysis-(seahorse)/basic
-procedures-to-run-an-xf-assay
Load compounds into the appropriate ports of a hydrated
sensor cartridge.
•Standard Assay - No additional injection
Port A: Oligomycin
Port B: FCCP
Port C: Rotenone/antimycin A
• Modified Assay - Additional injection included. To inject an
additional compound prior to oligomycin, use port A for the
desired compound and then load:
Port B: Oligomycin
Port C: FCCP
Port D: Rotenone/antimycin A
Tab le 5 Compound Preparation for Loading Sensor Cartridge Ports for
Seahorse XFe/XF 24 analyzers. Starting with well volume of
500 µL assay medium
(Final well)
(µM)
Stock
volume
(µL)
Media
volume
(µL)
10X (Port)
(µM)
Add to
port (µL)
Port A
Oligomycin
0.5 150 2,850 5 56
1.0 300 2,700 10 56
2.0 600 2,400 20 56
Port B
FCCP
0.125 37.5 2,962.5 1.25 62
0.25 75 2,925 2.5 62
0.5 150 2,850 5 62
1.0 300 2,700 10 62
2.0 600 2,400 20 62
Port C
Rotenone/
antimycin A
0.5 300 2,700 5 69
18 Agilent Seahorse XF Cell Mito Stress Test Kit User Guide
Assay Workflow
Table 6 lists the appropriate volumes and concentrations for
this injection scheme.
Prepare Agilent Seahorse XF Cell Culture Microplate for assay
1Remove Seahorse XF Cell Culture Microplates from 37 °C
CO2 incubator and examine the cells under a microscope to
confirm confluence.
2Remove the assay medium from water bath.
3Change the cell culture growth medium in the cell culture
microplate to warmed assay medium using a multichannel
pipette, and place the cell culture microplate into a 37 °C
non-CO2 incubator for 45 minutes to 1 hour prior to the
assay.
Running the Assay
Open Wave and retrieve saved assay template file. Follow the
instructions below:
If you are using Wave:
1Browse for and open the saved design file.
2Click Run.
3Place the calibration plate with the loaded sensor cartridge
on the instrument tray, and click Continue. Calibration takes
approximately 15-30 minutes.
Note: Remove the cartridge lid and verify correct plate
orientation.
4When prompted, replace the calibration plate with the cell
culture microplate then click Start.
Tab le 6 Compound Injection Volumes Involving an Acute Injection. For
Seahorse XFe/XF 96 analyzers, start with well volume of
180 µL assay medium. For Seahorse XFe/XF 24 Analyzers,
start with well volume of 500 µL assay medium
Port
Seahorse XFe/XF 96 Seahorse XFe/XF 24
Add to port
volume (µL)
Port
concentration
Add to port
volume (µL)
Port
concentration
A 20 10X 56 10X
B 22 10X 62 10X
C 25 10X 69 10X
D 27 10X 75 10X
Assay Workflow
Agilent Seahorse XF Cell Mito Stress Test Kit User Guide 19
If you are using Wave:
1Browse for and open the saved design file, select the Review
and Run tab, and then click Start Run.
2When prompted, place the loaded sensor cartridge with the
calibrant plate into the instrument, then click I'm ready.
Calibration takes approximately 15-30 minutes.
Note: Remove the cartridge lid and verify correct plate
orientation.
3Following calibration and equilibration of the cell culture
microplate, when prompted click I'm ready.
4Load the cell culture microplate, and click I'm ready to run
the assay.
Data Analysis
The Seahorse XF Mito Stress Test Report Generator
automatically calculates the Seahorse XF Cell Mito Stress Test
parameters from Wave data that has been exported to Excel.
The Seahorse XF Stress Test Report Generator can be used with
either a standard or modified stress test protocol, and provides
a convenient, customizable, one-page assay summary.
The Seahorse XF Report Generator can be installed either
alongside Wave or directly from the Seahorse Bioscience
website. Visit
http://www.agilent.com/en-us/support/cell-analysis-(seahorse)
/seahorse-xf-report-generators to learn more about the
Seahorse XF Stress Test Report Generators and download the
User Guide.
20 Agilent Seahorse XF Cell Mito Stress Test Kit User Guide
Assay Workflow
Agilent Technologies
© Agilent Technologies, Inc.
Printed in USA, March 2017
Revision E0
For research use only
Not for use in diagnostic procedures
*103016-400*
103016-400
