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Agilent Technologies, Inc.

Seahorse XF Substrate Oxidation Stress Test Kits

The Agilent Seahorse XF Substrate Oxidation Stress Test Kits are a suite of optimized solutions for rapidly measuring cellular substrate oxidation by assessing changes in oxygen consumption (OCR) in live cells. These kits allow for the interrogation of three primary substrates that fuel the mitochondria: long chain fatty acids , glucose/pyruvate, and/or glutamine, facilitating investigation of how cells alter or shift oxidation of specific substrates to perform key cellular functions of activation, proliferation, and differentiation, as well as to respond to genetic or pharmaceutical interventions or survival in microenvironments. By combining the well-recognized XF assay for mitochondrial function (Cell Mito Stress Test) with substrate pathway specific inhibitors (etomoxir, UK5099, BPTES), these kits report multiple functional parameters, providing rich and robust data. The kits are supplied with validated reagents, standardized protocols, and easy-to-use Seahorse Analytics to enhance the user experience.

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  • Discover how cell phenotype or fate can be controlled via manipulation or control of substrate oxidation activities
  • Understand which substrates are relevant or required for specific cellular function and/or phenotype using specific, optimized XF Substrate Oxidation Stress Test kits and protocols
  • Build on the well-accepted XF assay (XF Mito Stress Test) for interrogating mitochondrial metabolism: dive deeper into effects of specific substrate oxidation on cellular function, including activation, proliferation, differentiation, and dysfunction
  • Use specific substrate pathway inhibitors to reveal cells’ dependency on three primary mitochondrial substrates: long chain fatty acids, glucose/pyruvate, and/or glutamine under both basal respiration and high energy demand condition
  • May be used individually or in combination, allowing investigation of single or multiple substrates at the same time
  • Provides rich real-time multiparametric information not available from conventional endpoint substrate uptake or utilization assays
  • Compounds are reconstituted directly in the assay medium, eliminating the need for DMSO or other solvents
  • Every lot is rigorously tested and validated with functional assays to deliver consistent and robust results
  • Upload your assay result file to your Agilent Seahorse Analytics account to calculate and report parameters of the XF Substrate Oxidation Stress Test, share with collaborators, or export data to Microsoft Excel or GraphPad Prism for other graphing or analysis needs

How it works

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Rapid Seahorse XF methods that measure mitochondrial substrate oxidation

Long-chain fatty acids (LCFAs), glucose/pyruvate and glutamine are three primary substrates that fuel mitochondrial metabolism. Researchers in many areas (e.g., cancer, immunology, stem cell biology) have demonstrated how oxidization of these substrates has profound impact on cell fate, function, and fitness. Many of the findings not only provide new biological insights, but also reveal novel approaches to intervention and developing successful research therapies. The Seahorse XF Substrate Oxidation Stress Test Kits enable rapid assessment of substrate oxidation in live cells.

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Revealing substrate dependency under basal and high energy demand states

The assay involves an acute injection of a single pathway inhibitor (etomoxir, UK5099, or BPTES),  followed by 3 sequential injections of oligomycin, FCCP, and rotenone/antimycin A. This design provides information for basal respiration and acute response to the inhibitor (i.e., basal response), as well as maximal respiration, +/- the inhibitor. While minimal changes may be observed under basal conditions, larger responses are revealed in maximal respiration under high energy demand, which increases the dynamic window to measure changes in oxidation of specific substrates.

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Flexible assay design and more kit options to address different research needs

The kits can be used individually or in combination, allowing testing of a single substrate or multiple substrates in one experiment. The basic procedure is performed in standard assay conditions and is best used for examining how mitochondrial substrate demand and reliance is affected when an intervention (e.g., a genetic manipulation or drug exposure) is applied to the cells.  
Note that an advanced protocol and a kit designed for explicit investigation of long-chain FAO using palmitate-BSA are also available. For details, visit the Agilent website for product 103693-100.



Download the Agilent Seahorse XF Assay Kits & Reagents Overview
Download the new Media Selection Guide
Download the Seahorse Plastics Brochure
Download the Seahorse XF Instrument Selection Guide


Agilent Technologies, Inc.

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