Agilent XF Substrate Oxidation Stress User Manual

Agilent XF Substrate Oxidation Stress Test Kits

User Manual

XF Long Chain Fatty Acid Oxidation Stress Test Kit (103672-100) XF Glucose/Pyruvate Oxidation Stress Test Kit (103673-100) XF Glutamine Oxidation Stress Test Kit (103674-100) XF Palmitate Oxidation Stress Test Kit (103693-100)

Notices

CAUTION

WARNING

No part of this manual may be reproduced in any form or by any means (including electronic storage and retrieval or translation into a foreign language) without prior agreement and written consent from Agilent Technologies, Inc. as governed by United States and international copyright laws.

Manual Part Number

5994-1164EN Rev B0

Edition

First edition, May 2020

Printed in USA

Agilent Technologies 1834 Hwy 71 West  Cedar Creek, TX 78612

Product is made in UK.

Warranty

The material contained in this document is provided “as is,” and is subject to being changed, without notice, in future editions. Further, to the maximum extent permitted by applicable law, Agilent disclaims all warranties, either express or implied, with regard to this manual and any information contained herein, including but not limited to the implied warranties of merchantability and fitness for a particular purpose. Agilent shall not be liable for errors or for incidental or consequential damages in connection with the furnishing, use, or performance of this document or of any information contained herein. Should Agilent and the user have a separate written agreement with warranty terms covering the material in this document that conflict with these terms, the warranty terms in the separate agreement shall control.

Technology Licenses

The hardware and/or software described in this document are furnished under a license and may be used or copied only in accordance with the terms of such license.

Restricted Rights Legend

U.S. Government Restricted Rights. Software and technical data rights granted to the federal government include only those rights customarily provided to end user customers. Agilent provides this customary commercial license in Software and technical data pursuant to FAR 12.211 (Technical Data) and

12.212 (Computer Software) and, for the Department of Defense, DFARS

252.227-7015 (Technical Data -Commercial Items) and DFARS 227.7202-3 (Rights in Commercial Computer Software or Computer Software Documentation).

Safety Notices

A CAUTION notice denotes a  hazard. It calls attention to an operating procedure, practice, or the like that, if not correctly performed or adhered to, could result in damage to the product or loss of important data. Do not proceed beyond a CAUTION notice until the indicated conditions are fully understood and met.

A WARNING notice denotes a hazard. It calls attention to an operating procedure, practice, or the like that, if not correctly performed or adhered to, could result in personal injury or death. Do not proceed beyond a WARNING notice until the indicated conditions are fully understood and met.



1Introduction

Assay Background 6

XF Substrate Oxidation Stress Test - Standard Assay 8

XF Palmitate Oxidation Stress Test - Advanced Assay 12

Glossary 16

References 18

2 Kit Information

Kit Contents 20

Kit Shipping and Storage 21

Additional Agilent Products Required 21

3 Assay Workflow for Standard Substrate Oxidation Stress Test

One Day Prior to Assay 24

Day of Assay 24

Data Analysis Using Agilent Seahorse Analytics 30

Examples of Data 32

4 Assay Workflow for Palmitate Oxidation Stress Test - Advanced Assay

Two Days Prior to Assay 36

One Day Prior to Assay 36

Day of Assay 37

Data Analysis Using Agilent Seahorse Analytics 42

Examples of Data 44

5 Frequently Asked Questions

6 Context of Substrate Oxidation Tests Among Other XF Kits and Applications

Agilent XF Substrate Oxidation Stress Test Kits 3



4 Agilent XF Substrate Oxidation Stress Test Kits

1 Introduction

Assay Background 6

XF Substrate Oxidation Stress Test - Standard Assay 8

XF Palmitate Oxidation Stress Test - Advanced Assay 12

Glossary 16

References 18

Agilent XF Substrate Oxidation Stress Test Kits 5

Assay Background

MPC

Glucose

ATP

Glucose

Glycolysis

Glutamate

Malate

Acetyl-CoA

Ⱥ-KG

ADP + Pi

ETC

Acyl-

CoA

TCA

cycle

Amino

acids

UK5099

Pyruvate

Glutamine

LCFAs

BPTES

Eto

Gluconeogenesis

LCFAs

Glutamine

CPT1a

CPT2

Ȼ-Oxidation

GLS1

Gln transporter

Lactate

Assay Background

Agilent Seahorse XF technology measures energy metabolism in live cells in real time, providing critical functional information that relates directly to cellular health and fitness. The Seahorse XF Substrate Oxidation Stress Test Assay provides key metrics to facilitate the assessment of specific mitochondrial substrates that are relevant or required for cellular phenotype and function.

Agilent provides a suite of optimized, rapid solutions for measuring cellular substrate oxidation by assessing changes in oxygen consumption (OCR) using live cells. XF Substrate Oxidation Stress Test Kits allow for both a sensitive measure of mitochondrial function, and the interrogation of three primary substrates that fuel mitochondria: long-chain fatty acids (LCFAs), glucose/pyruvate (G/P), and/or glutamine (Q) (Figure 1). These kits facilitate the convenient investigation of specific substrate oxidation processes and the central role they play in the fundamental cellular functions of activation, proliferation, and differentiation; as well as in better characterizing cellular responses to genetic manipulation, pharmaceutical interventions, or specific disease-relevant microenvironments.

6 Agilent XF Substrate Oxidation Stress Test Kits

Figure 1. Primary metabolic pathways, including glycolysis, the TCA cycle, electron transport (ETC),

and oxidative phosphorylation (OXPHOS). Glucose/pyruvate, glutamine and long chain fatty

The XF Substrate Oxidation Stress Tests combine the substrate pathway specific inhibitors (Figure 1): etomoxir for LCFAs through inhibition of carnitine palmitoyl transferase 1a (CPT1a) UK5099 for glucose and/or pyruvate through inhibition of the mitochondrial pyruvate carrier (MPC) Mito Stress Test (MST). The MST, a powerful and well-accepted tool for the interrogation of mitochondrial function, in conjunction with these inhibitors, can be used to reveal dependence on a specific metabolic substrate. Basal and maximal respiration rates are key metrics of

acid oxidation are highlighted. Red lines denote relevant inhibitors of glucose/pyruvate, LCFA, and glutamine transport, which in turn, specifically limits oxidation of that respective substrate.

; and BPTES for inhibition of glutamine through glutaminase 1 (GLS-1)3, with the XF Cell

;

Assay Background

mitochondrial function reported by the MST. In the context of substrate oxidation, the basal, and in particular, the maximal respiration rates are largely impacted by cells capacity to transport and oxidize available substrates.

This method is ideally suited to the assessment for cellular substrate demand both under basal conditions, and in response to elevated substrate demand (maximal respiration). Figure 2 outlines the kinetic profile of a standard substrate oxidation assay and relevant assay parameters.

Figure 2. Agilent Seahorse XF Substrate Oxidation Stress Test profile of the respiration parameters

critical for substrate demand. Sequential compound injections measure basal respiration, acute response to an inhibitor (etomoxir or UK5099 or BPTES), and maximal respiration in the absence and presence of inhibitor. Note that while minimal changes may be measured under basal conditions, such as the acute response; much larger responses are often revealed under conditions of high substrate demand (for example, FCCP), thus revealing differences in the ability of the cells to oxidize the substrate in question.

Measurements are highly informative, as the design described provides information on basal respiration and the impact of pathway inhibition under conditions of basal substrate demand, while also characterizing the impact of pathway inhibitions on maximal respiration, reflecting the cell's sensitivity to impairment of a specific metabolic pathway under conditions of high substrate demand. Due to the metabolic plasticity of many cell types, it is often more informative to assess substrate dependence when demand is high as this reveals the cell's capacity to use substrates. When mitochondria are uncoupled (such as FCCP exposure), substrate oxidation is increased to generate the reducing equivalents needed for the increased respiration rate. By comparing the maximum respiration rates of control versus inhibited cells, insights can be gained into how much a cell will rely on a specific substrate to meet this high energy demand.

This manual details how to perform all assays in the XF Substrate Oxidation Stress Test suite. In general, there are two distinct assay types: Standard and Advanced. The XF LCFA Oxidation Stress Test, XF Glutamine Oxidation Stress Test and Glucose/Pyruvate Oxidation Stress Test kits are to be carried out using the Standard Assay workflow, using a single inhibitor in the presence of multiple substrates). Conversely, the XF Palmitate Oxidation Stress Test Kit requires users to use the Advanced Assay protocol as additional steps are required (Figures 5, 6, and 10), and is carried out using palmitate as the long chain fatty acid substrate and etomoxir as the inhibitor. The following are the principles and designs of these assays.

Agilent XF Substrate Oxidation Stress Test Kits 7

XF Substrate Oxidation Stress Test - Standard Assay

NOTE

XF Substrate Oxidation Stress Test - Standard Assay

Relevant kits include:

XF Long Chain Fatty Acid Oxidation Stress Test Kit (p/n 103672-100) XF Glucose/Pyruvate Oxidation Stress Test Kit (p/n 103673-100) XF Glutamine Oxidation Stress Test Kit (p/n 103674-100)

To perform a Standard Substrate Oxidation Stress Test, basal respiration is first established, followed by injection of the relevant pathway inhibitor. The acute response to the inhibitor is monitored over several measurement cycles (typically six). Then, the standard XF Cell Mito Stress Test reagents, such as oligomycin, FCCP, and rotenone/antimycin A are injected sequentially, similar to the assay scheme used for the XF Cell Mito Stress Test. Figure 3 outlines an overview of the experimental workflow for each kit.

Figure 3. Agilent XF Substrate Oxidation Stress Test - Standard Assay Design. Each kit is focused on testing a single substrate

using the relevant inhibitor. Chronic or acute interventions (genetic manipulations/drug exposure) may be performed upstream of the assay to understand the effects of these modulations on the oxidation of specific mitochondrial substrates.

Each kit is focused on testing a single substrate using the optimized concentration of relevant inhibitor: etomoxir (4 µM) to inhibit oxidation of LCFAs, UK5099 (2 µM) to inhibit the oxidation of glucose and/or pyruvate, and BPTES (3 µM) to inhibit oxidation of glutamine (final concentrations). These three assays are designed to be performed under conditions of saturating substrates with respect to glucose (10 mM), pyruvate (1 mM) and glutamine (2 mM) in the assay media. The source of long chain fatty acids is any endogenous stores of lipid/LCFAs in the cell used, and is cell-type dependent. Figure 3 shows that the standard methods and assay conditions for each of the XF Substrate Oxidation Stress Tests are identical with the exception of the identity of the inhibitor used.

Users must first establish both the optimal cell seeding density and optimal FCCP concentration for ideal assay performance and resulting data. Typically, these are the same conditions established for a cell type in an XF Cell Mito Stress Test. For information on how to optimize cell seeding density and FCCP concentrations, please visit the Agilent Cell Analysis

Learning Center website.

8 Agilent XF Substrate Oxidation Stress Test Kits

XF Substrate Oxidation Stress Test - Standard Assay

In these Standard Assays, decreased respiration rates in response to an inhibitor suggest that the cells have a demand or preference for that particular substrate under the experimental conditions established. In general, these assays can be used to facilitate investigation to address the following types of questions:

• Does the cell have a demand for a particular substrate or substrates?

• Is the cell highly reliant on a specific substrate, or can other substrates satisfy cellular

demands?

• How is mitochondrial substrate demand and/or reliance affected if an intervention, such as

genetic manipulation or drug exposure, is applied to the cell?

Like many XF assays, the Substrate Oxidation Stress Tests are typically performed subsequent to a pretreatment, or intervention, as designed by the researcher. This is shown as either a chronic intervention to the cells (for example, a genetic manipulation or long-term drug exposure), hours to days upstream of XF assay, or as an acute intervention (for example, drug exposure) just prior to the XF assay (Figure 3). In some cases, both chronic and acute interventions may be used (for example, rescue of genetic dysfunction through compound exposure). The kits may be used individually (such as focusing on one specific substrate) for investigating how a series of interventions or compounds may affect oxidation of that particular substrate; or in combination (such as focusing on two or more substrates) to elucidate overall effects of a given intervention with respect to substrate oxidation and mitochondrial function.

For examples of the design of assay templates for XF Substrate Oxidation Stress Tests - Standard Assays, refer to Figure 4.

Agilent XF Substrate Oxidation Stress Test Kits 9

XF Substrate Oxidation Stress Test - Standard Assay

10 Agilent XF Substrate Oxidation Stress Test Kits

XF Substrate Oxidation Stress Test - Standard Assay

Figure 4. Agilent Seahorse XF Substrate Oxidation Stress Test - Assay Design Examples. These

examples are suggested designs, and the user is encouraged to modify the generic Standard Substrate Oxidation Stress Test Assay Template file to accommodate the specific requirements of the experimental design.

Agilent XF Substrate Oxidation Stress Test Kits 11

XF Palmitate Oxidation Stress Test - Advanced Assay

XF Palmitate Oxidation Stress Test - Advanced Assay

Relevant kit includes:

XF Palmitate Oxidation Stress Test Kit (p/n 103693-100)

The XF Palmitate Oxidation Stress Test Advanced Assay details the workflow to specifically analyze the long chain fatty acid oxidation pathway in live cells. The kit includes the XF Palmitate-BSA FAO Substrate, L-carnitine, etomoxir, as well as oligomycin, FCCP, and rotenone/antimycin A, and is designed to determine the intrinsic rate and capacity of a cell to oxidize palmitate in the absence or limitation of other exogenous substrates. This workflow is best applied when investigating how interventions (genetic manipulations and drug exposure) specifically affect the LCFA oxidation process and can be a complementary and/or follow-up assay to the Standard Assay workflow for XF Long Chain Fatty Acid Oxidation Stress Test discussed in the previous section. The kinetic profile of the Advanced Palmitate oxidation assay and relevant assay parameters is outlined in Figure 5, and an overview of the experimental workflow for the Advance Assay is outlined in Figure 6.

Figure 5. Agilent Seahorse XF Palmitate Oxidation Stress Test profile of the respiration parameters

critical for palmitate demand. Sequential compound injections measure basal respiration, acute response to etomoxir, and maximal respiration in the absence and presence of etomoxir. Note that while minimal changes may be measured under basal conditions, such as the acute response, much larger responses are often revealed under conditions of high substrate demand (for example, FCCP), revealing differences in the ability of the cells to oxidize palmitate.

12 Agilent XF Substrate Oxidation Stress Test Kits

XF Palmitate Oxidation Stress Test - Advanced Assay

NOTE

Figure 6. Agilent XF Palmitate Oxidation Stress Test - Advanced Assay. This assay is specifically focused on determining the

intrinsic rate and capacity of a cell to oxidize palmitate in the absence or limitation of other exogenous substrates, using inhibitor etomoxir to inhibit oxidation of palmitate.

The Palmitate Oxidation Stress Test is designed to be performed under conditions of saturating palmitate (as palmitate-BSA) and L-carnitine, limited to no substrates provided with respect to glucose, pyruvate, and glutamine in the assay media. It is best applied when the experimental design calls for the cells to exclusively oxidize a long chain fatty acid, such as palmitate, to investigate effects of interventions specifically on the long chain fatty acid oxidation process.

As shown in Figure 6, this workflow often requires substrate limitation before and/or during the assay to condition the cells to obtain robust responses to the palmitate substrate added to the assay media. The suggested initial substrate-limited growth media conditions can be found in

Table 1.

Users must first establish the optimal cell density and optimal FCCP concentration for best assay performance and resulting data. Typically, these are the same conditions established for a cell type in an XF Cell Mito Stress Test, except the FCCP concentration may need to be re-optimized in the presence of BSA. For information on how to optimize cell density and FCCP concentrations, please visit the Agilent Cell Analysis Learning Center website.

Table 1 Suggested substrate limitation conditions for performing the XF Palmitate Oxidation

Stress Test Advanced Assay with base growth media. Initial suggested time of incubation under substrate limitation is overnight (16 to 24 hours).



Base growth media

DMEM or RPMI  without glucose, pyruvate,  glutamine, or GlutaMAX



Growth media supplement

Glucose 0.5 mM

Glutamine or GlutaMAX 1.0 mM

Serum (for example, FBS) 1%

Seahorse L-Carnitine 0.5 mM

Suggested initial concentration in substrate-limited growth media

Table 2 Suggested substrate limitation conditions for performing the XF Palmitate Oxidation

Stress Test Advanced Assay with XF Assay Media.

XF Assay Media Assay Media supplement Suggested initial concentration in assay media

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

XF Glucose 2.0 mM

XF L-Carnitine 0.5 mM

XF Palmitate-BSA

96 well 30 µL/well

24 well 85 µL/well

XF BSA Control

* Note that XF Palmitate-BSA and XF BSA control are added directly to the XF cell culture plate wells just prior to starting the

assay. See pages 37 - 38 for further information.

96 well 30 µL/well

24 well 85 µL/well

Agilent XF Substrate Oxidation Stress Test Kits 13

XF Palmitate Oxidation Stress Test - Advanced Assay

NOTE

Optimal limited substrate concentrations and optimal time of incubation are cell-dependent and should be empirically determined for the cell type of interest.

Instead of examination of substrate demand and reliance, this Advanced Assay is designed to be used when asking the following type of question:

• How an intervention, such as genetic manipulation or drug exposure, specifically affects the

oxidation of long chain fatty acids when applied to the cell?

This assay is most often performed under some pretreatment condition, or intervention, as designed by the researcher. This is shown as either a chronic intervention to the cells (for example, a genetic manipulation or long-term drug exposure), hours to days upstream of XF assay, or as an acute intervention (for example, drug exposure) just prior to the XF assay (Figure 6). In some cases, both chronic and acute interventions may be used (for example, rescue of genetic dysfunction through compound treatment). For examples of the assay template design for XF Palmitate Oxidation Stress Test - Advanced Assay, refer to Figure 7.

There are critical and distinct difference between this Advanced Assay workflow and the Standard Assay workflow described in the previous section, including:

• Cells usually require a period of substrate limitation to measure oxidation of palmitate

substrate provided, especially established cell lines. The specific conditions will be cell type dependent, and should be determined empirically for each cell type tested, particularly for primary type cells.

• An analogous BSA control group must be included for each condition tested to ensure that the

observed responses are associated with Palmitate-BSA in the assay media. Therefore, the number of groups used doubles compared to a Standard Substrate Oxidation Stress Test. For examples of Palmitate Oxidation Stress Test assay templates, refer to Figure 6.

• L-carnitine is included in substrate-limited medium and assay medium to ensure that it is not a

rate limiting factor in the assay.

• The conditions for substrate limitation and performance of the Advanced Assay are only

validated for use with the XF Palmitate-BSA substrate and BSA control.

14 Agilent XF Substrate Oxidation Stress Test Kits

XF Palmitate Oxidation Stress Test - Advanced Assay

Figure 7. Agilent XF Palmitate Oxidation Stress Test - Plate Layout Map Examples.

Agilent XF Substrate Oxidation Stress Test Kits 15

Glossary

Glossary

• Basal Respiration: Oxygen consumption used to meet cellular ATP demand and resulting from

mitochondrial proton leak. Shows substrate demand of the cell under baseline conditions.

• Acute Response: Change in oxygen consumption rate due to an injection of substrate

oxidation inhibitor (etomoxir, UK5099, BPTES). Reported as a change in OCR (i.e., ∆OCR pmol/min).

• Maximal Respiration: The maximal oxygen consumption rate attained by adding the

uncoupler FCCP. FCCP increases a substrate demand by stimulating the respiratory chain to operate at maximum capacity, which causes rapid oxidation of substrates (sugars, fats, and amino acids) to meet this metabolic challenge. Shows substrate demand of the cell under maximal respiration conditions.

• Non-mitochondrial Respiration: Oxygen consumption that persists due to a subset of cellular

enzymes that continue to consume oxygen after rotenone/antimycin A addition. This is important for getting an accurate measure of mitochondrial respiration.

• Standard Substrate Oxidation Stress Test Workflow: Three complementary assays, each

focused on testing cellular demand of a single substrate (LCFAs or G/P or Q) using the optimized concentration of relevant inhibitor: etomoxir or UK5099 or BPTES. Designed to be performed under conditions of saturating substrates with respect to glucose, pyruvate, and glutamine in the assay media (Figures 2, 3, 4, and 8).

• Advanced XF Palmitate Oxidation Stress Test: A single assay designed to determine the

intrinsic rate and capacity of a cell to oxidize palmitate in the absence or limitation of other exogenous substrates. Performed under conditions of saturating palmitate (as palmitate-BSA) and L-carnitine, in the assay media. It is best applied when the experimental design calls for the cells to exclusively oxidize a long-chain fatty acid, such as palmitate, in order to investigate effects of interventions specifically on the long chain fatty acid oxidation process (Figures 5, 6, 7, 12, and Tab l e 1).

•MST: XF Cell Mito Stress Test, a well-recognized assay that provides a comprehensive view of

mitochondrial function by reporting multiple parameters for mitochondrial respiration. For more information, visit the Agilent Seahorse XF Cell Mito Stress Test product website.

•FAO: Fatty acid oxidation.

•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. Note that concentrations in excess of 4 µM (final in assay) result in substantial off-target effects on mitochondrial respiration

• UK5099: An inhibitor of the glucose oxidation pathway. UK5099 inhibits the mitochondrial

pyruvate carrier (MPC), which transports pyruvate into the mitochondria. 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. Note that BPTES does not inhibit GLS2.

•LCFAs: Long chain fatty acids.

•G/P: Glucose/pyruvate.

•Q: Glutamine.

16 Agilent XF Substrate Oxidation Stress Test Kits

Glossary

• L-Carnitine: A supplement provided in the Palmitate Oxidation Stress Kit for use in substrate

limited growth media and substrate limited assay media. Ensures concentrations of L-carnitine are saturating and not limiting rates of palmitate oxidation.

•Growth Media: Fully supplemented cell culture media appropriate for the specific cell type.

• Substrate Limitation: A condition used in the Palmitate Oxidation Stress Test - Advanced

Assay. It usually refers to a period of time (e.g., overnight) in which key substrates (typically glucose, pyruvate, GlutaMAX, and serum) are reduced in concentration in the cell culture media.

• Substrate-limited growth media: Cell culture media with key substrates (typically glucose,

pyruvate, GlutaMAX, and serum) reduced in concentration (see Table 1).

• Substrate-limited assay media: Assay media with key substrates (typically glucose, pyruvate,

and glutamine) reduced in concentration and/or omitted (see Table 2).

Agilent XF Substrate Oxidation Stress Test Kits 17

+ 37 hidden pages