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Agilent Technologies, Inc.

Seahorse XF Mito Tox Assay Kit

The Agilent Seahorse XF Mito Tox Assay kit allows for the identification of drug-induced mitochondrial toxicity through direct functional measurements of mitochondrial oxygen consumption rates (OCR). This kit is adapted from the Agilent Seahorse XF Cell Mito Stress Test, the fundamental assay for interrogating mitochondrial function, with improved assay design and data outputs. It provides high sensitivity and specificity along with the toxicity mode of action in one assay, minimizing false positives and false negatives. The XF Mito Tox Assay Kit seamlessly integrates with Agilent Seahorse Pro Controller and Wave Pro software and Agilent Seahorse Analytics in the assay workflow, providing an end-to-end solution for assessing drug-induced mitochondrial toxicity. The software features greatly simplify the entire XF assay experience, from assay design or template creation to data interpretation, facilitating decision making to de-risk drug pipelines.

Each assay kit contains sufficient materials for six full-plate tests in 96-well format. The assay is only compatible with the Agilent Seahorse XF Pro analyzer.

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  • High sensitivity and specificity for identification of mitochondrial toxicity through direct measurement of mitochondrial dysfunction
  • Information rich data allowing for distinguish inhibitors and uncouplers in one assay
  • Improved assay robustness and throughput through the new features of the XF Pro analyzer, consumables, and assay design
  • Enhanced usability in assay template creation, data quality assessment, and data reporting process
  • Dedicated widgets in Seahorse Analytics that reports Mito Tox Indices (MTI), IC50/EC50, and Z’ automatically
  • Validated for use with Agilent Seahorse XF Pro analyzer and XF Pro M plate. Best performed with Agilent Seahorse XF DMEM medium, pH 7.4 (p/n 103575-100) and supplements (p/n 103577-100, 103578-100, 103579-100)
  • 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
  • Use this link to access Seahorse Analytics for data analysis: https://seahorseanalytics.agilent.com

How it works

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Mitochondrial Toxicity and Drug Safety

Mitochondria are the powerhouses of cells and are involved in numerous cellular functions. Disruption to mitochondrial function can be detrimental and is linked to drug-induced liver injuries, cardiac and neurological damages and more, leading to drug attrition and postmarket drug withdrawals. Early identification of compound perturbation to mitochondrial function, by incorporating mitochondrial toxicity test into drug safety assay panel, allows for optimization of compound chemistry upstream, avoiding drug attrition in later development stages and derisking drug pipelines.

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XF Mito Tox Assay Design and Workflow

The XF Mito Tox assay is adapted from the XF Cell Mito Stress Test. This refined assay includes sequential injections of oligomycin and FCCP, with a separate experimental group pre-exposed to Rotenone/antimycin A. This design produces robust assay windows to deliver industry-accepted assay performance (Z’>0.5). When used with enhanced software tools, the assay reports Mito Tox Index (MTI), which identifies inhibitors and uncouplers in one assay. Short- or long-term toxic effects can be assessed through a pre-incubation with the test compounds prior to performing the assay.

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Application in Screening and Dose-Response Assays

The XF Mito Tox assay workflow can be used to perform screening assays at a single dose with a throughput of 80 compounds per plate. The raw data are automatically transformed into organized and sharable reports by Seahorse Analytics. Hits can be presented in plate heat maps or bar charts in the desirable orders. The workflow is also applicable to dose-response experiments to identify IC50/EC50. Performance metric (Z’) is reported for every assay. This workflow greatly simplifies the entire assay experience, from assay design (template creation) to data interpretation.



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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Measure mitochondrial respiration and glycolysis as well as ATP production rate of live cells.

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