Agilent Seahorse XF Palmitate-BSA FAO
Substrate Quickstart Guide
For use with Agilent Seahorse XFe, XF, and XFpAnalyzers
Product Description 2Introduction 3
Medium and Buffer Preparation 4
Running Fao Assay 5
References 7
Product Use and Limitations, Warranty, Disclaimer 8

Kit part number: 102720-100

Agilent Technologies

Product Description
The Agilent Seahorse XF Palmitate-BSA fatty acid oxidation (FAO) Substrate isa bio-available palmitate reagent for researchers studying substrate utilization
who require a reliable source of conjugated fatty acids. The XF Palmitate-BSA
FAO Substrate is manufactured at a 6:1 Palmitate:BSA ratio. Each lot is tested
for activity and free fatty acid (FFA) concentration.

Contents• 1 mM palmitate conjugated to 0.17 mM BSA in 150 mM NaCl, pH 7.2,
3 × 2 mL vials• 0.17 mM BSA control in 150 mM NaCl, pH 7.2, 3 × 2 mL vials. Lot-specific
FFA is listed on each box

Storage conditions
Upon arrival, store at -20 °C, aliquot and refreeze if desired. Glass vials arerecommended for storage. The stability of the product is still under evaluation,
but has a minimum 6-month shelf life when properly stored. Do not store in a
frost-free freezer.

Reagent preparation• Thaw solution at room temperature.
• Prior to beginning the assay, add undiluted substrate or control directly tothe well. We recommend adding the substrate directly to the microplate
instead of to the injection ports to avoid inconsistent injection performancedue to viscosity.
Table 1

2

Recommended volumes: Palmitate-BSA or BSA control

Component

VolumeXFe24/XF24

87.5 µL

XFe96/XF96

30 µL

Seahorse XF Palmitate-BSA FAO Substrate Quickstart Guide

IntroductionThe ability to oxidize exogenously added fatty acids (FAs), as indicated by anincrease in O2 consumption rate (OCR), will be influenced by ATP demand,
other substrates present in the assay medium (such as glucose), and stores ofendogenous substrates (such as glycogen or triglycerides). Exogenously added
FAs can also uncouple mitochondria, and cause an increase in the OCR not
due to fatty acid oxidation (FAO).
Palmitate-BSA has been used extensively to examine FA oxidation. The
addition of etomoxir to a cell, and the subsequent return to the control rate ofrespiration, demonstrate utilization of exogenous fatty acids1,2. A drop in OCR
in both the palmitate and BSA control indicates that endogenous FAs werealso being oxidized3. Finally, if the OCR signal is insensitive to etomoxir oroligomycin, this indicates that the FAs are uncoupling the mitochondria4.
Because a change in OCR can be caused by utilization of exogenous FAs,endogenous FAs, or uncoupling by FAs, Agilent Technologies developed the XF
FAO Assay that can measure all three sources. With the combined use of theXF Palmitate-BSA FAO Substrate, etomoxir, and the XF Cell Mito Stress Test,
one can drive cells to oxidize exogenous fatty acids while revealing the portionof the OCR signal generated by endogenous and exogenous fatty acidoxidation, as well as uncoupling.
The assay requires components that are not included with the this product,
which is referenced in Table 2. A guideline on how to perform FAO assay isprovided in this document. This assay was developed using immortalized celllines. Use of primary cultures may require optimization of reduced nutrient
conditions.
Table 2Items required but not provided

Component

Details

XFe24/96 or XF24/96 Analyzer

www.agilent.com/en-us/products/cell-analysis-(seahorse)

XFe/XF FluxPak or FluxPak Mini

www.agilent.com/en-us/products/cell-analysis-(seahorse)

Substrate-Limited Medium

See formulation below

FAO Assay Medium

See formulation below

Etomoxir (10 mM Stock Solution in H2O)

Sigma-Aldrich, catalog #E1905, (+)-etomoxir sodium salt hydrate

XF/XFp Cell Mito Stress Test Kit

Agilent Technologies, catalog #103015-100, #103010-100

Seahorse XF Palmitate-BSA FAO Substrate Quickstart Guide3Medium and Buffer Preparation
Table 3

Substrate-limited medium

Component

Details

DMEM

Corning #17-207-CV or Life Technologies catalog #A14430-01 are DMEM
formulations without glucose, glutamine, sodium pyruvate, or HEPES

0.5 mM Glucose

D-(+)-Glucose, catalog #G7021, Sigma-Aldrich

1.0 mM GlutaMAX

GlutaMAX Supplement, catalog #35050061, Life Technologies

0.5 mM Carnitine

L-Carnitine hydrochloride, Sigma-Aldrich, catalog #C0283

1 % FBS

Fetal Bovine Serum

Table 4

FAO Assay buffer

Component

1x Concentration (mM)

5x Concentration (mM)

NaCl111555

KCl4.723.5CaCl2*

1.25

6.25

MgSO4

2.0

10NaH2PO4

1.2

6Supplements

On the day of the assay, supplement 1x KHB with 2.5 mM glucose, 0.5 mM carnitine and 5 mM
HEPES (final concentrations). Warm to 37 °C, and adjust pH to 7.4 with NaOH.

Carnitine may be purchased as L-carnitine hydrochloride from Sigma. Make as a 0.5 M stock solution in H2O and adjust pHto 7.2 with NaOH.
* This document was updated to include CaCl2 in the FAO Assay Medium

4Seahorse XF Palmitate-BSA FAO Substrate Quickstart Guide

Running Fao AssayNote: The XF assay work flow deviates from standard XF assays, and includes
two pretreatment steps:• Selected groups are treated with etomoxir 15 minutes prior to the XF assay
being initiated (t = –15 minutes).
• Selected groups are provided BSA control or Palmitate:BSA prior to the XF
assay being initiated (t = 0).

FAO Assay Workflow

Day 1

Day 2: Perform XF Assay

Exchange growth
medium for
substrate-limited
medium. Incubate
overnight.

t = –45 minutes
Exchange substratelimited medium for
FAO medium.

Figure 1

FAO Assay workflow.

Table 5

Recommended volumes

t = –15 minutes
Add etomoxir(40 µM ﬁnal)

t = 0 minutes
Add Palmitate:BSAor BSA control.
Initiate XF assay.

Conduct XF
Cell MitoStress Test.

Recommended volumes

XFe/XF24 (µL)

XFe/XF96 (µL)

Starting volume

375

135

400 µM Etomoxir (40 µM final)

37.5

15

BSA control or Palmitate:BSA

87.530

XF Cell Mito Stress Test Injections (all 10X) - final concentration
Port A: 25 µg/mL Oligomycin (2.5 µg/mL final) 50

20Port B: FCCP (TBD by user*)55

22

Port C: 20 µM Rotenone/40 µM Antimycin A(2 and 4 µM final, respectively)

60

24* Determination of maximal FCCP stimulation (maximal exogenous FA oxidation) should be conducted byperforming an FCCP titration in the context of the FAO assay workflow (that is, cells should be grown insubstrate-limited medium, assayed in FAO assay media, and be FCCP titrated in the presence of BSA Controland Palmitate-BSA).

Seahorse XF Palmitate-BSA FAO Substrate Quickstart Guide5

Determination of Fatty Acid Utilization in the XFe/XF Analyzer
Oligomycin

160

FCCP Rtn/AABSA, –EtoBSA, +Eto
Palm:BSA, –Eto
Palm:BSA, +Eto

OCR (pmol/min)

140120100

Oxidation of exogenous palmitate

8060402000Figure 210203040 50
Time (min)60708090

Kinetic graph of the FAO/XF Cell Mito Stress Test assay. The green arrow
indicates utilization of exogenous fatty acids. Note: in this case, utilization ofexogenous fatty acids was dependent on placing energetic stress (via FCCP) onthe cells. This experiment illustrates that under conditions of substrate
limitation, and the combined use of the XF Palmitate-BSA FAO Substrate,etomoxir, and the XF Cell Mito Stress Test, one can determine the proportion ofrespiration that is supported by exogenous fatty acids.

160
BSA, –Eto
BSA, +Eto
Palm:BSA, –Eto
Palm:BSA, +Eto

OCR (pmol/min)

140
120
100
80
60
40
20

6Eto,–SASAPalm:B:Blm
PaFigure 3,+EtooEt,+BSA

BS

A,–Eto0Bar chart highlighting the differences in maximal respiration. Data taken fromthe rate 10 measurement in Figure 2. The use of the XF Cell Mito Stress Test canelucidate different metabolic parameters allowing the determination if the cells
are oxidizing the exogeneous palmitate, as well as oxidizing endogenous fatty
acids or causing uncoupling.

Seahorse XF Palmitate-BSA FAO Substrate Quickstart GuideReferences
1 Ferrick, D. A.; et al. Advances in measuring cellular bioenergetics using

extracellular flux. Drug Discov. Today 2008, 13(5-6), 268-74.
2 Rowe, G. C.; et al. Disconnecting mitochondrial content from respiratory

chain capacity in PGC-1 deficient skeletal muscle. Cell Rep. 2013, 3(5),1449-1456.
3 Canto, C.; et al. AMPK regulates energy expenditure by modulating NAD+

metabolism and SIRT1 activity. Nature 2009, 458(23), 1056-1062.
4 Divakaruni, A. S.; Brand, M. D. The regulation and physiology ofmitochondrial proton leak. Physiology 2011, 26, 192-205.

Seahorse XF Palmitate-BSA FAO Substrate Quickstart Guide7

Agilent Technologies

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For Research Use Only. Not for use in diagnostic procedures.
This information is subject to change without notice.

© Agilent Technologies, Inc. 2017
Published in the USA, March 2017
Revision A0*102720-100*
102720-100