Related Experiment Video
Updated: Apr 21, 2026
Exploring Mitochondrial Energy Metabolism of Single 3D Microtissue Spheroids Using Extracellular Flux Analysis
Published on: February 3, 2022
The acute extracellular flux (XF) assay to assess compound effects on mitochondrial function
Ruolan Wang1, Steven J Novick2, James B Mangum2
1GlaxoSmithKline, Research Triangle Park, NC, USA ruolan.h.wang@gsk.com.
Abstract:
Numerous investigations have linked mitochondrial dysfunction to adverse health outcomes and drug-induced toxicity. The pharmaceutical industry is challenged with identifying mitochondrial liabilities earlier in drug development and thereby reducing late-stage attrition. Consequently, there is a demand for reliable, higher-throughput screening methods for assessing the impact of drug candidates on mitochondrial function. The extracellular flux (XF) assay described here is a plate-based method in which galactose-conditioned HepG2 cells were acutely exposed to test compounds, then real-time changes in the oxygen consumption rate and extracellular acidification rate were simultaneously measured using a Seahorse Bioscience XF-96 analyzer. The acute XF assay was validated using marketed drugs known to modulate mitochondrial function, and data analysis was automated using a spline curve fitting model developed at GlaxoSmithKline. We demonstrate that the acute XF assay is a robust, sensitive screening platform for evaluating drug-induced effects on mitochondrial activity in whole cells.
Insights
This study introduces a new assay to quickly screen drugs for mitochondrial toxicity. The extracellular flux (XF) assay helps identify potential drug liabilities early in development, improving safety.
Area of Science:
- Biochemistry and Molecular Biology
- Pharmacology and Toxicology
- Drug Development and Screening
Background:
- Mitochondrial dysfunction is linked to adverse health outcomes and drug toxicity.
- Early identification of mitochondrial liabilities in drug candidates is crucial for pharmaceutical development.
- There is a need for high-throughput screening methods to assess drug impact on mitochondrial function.
Purpose of the Study:
- To present and validate a novel extracellular flux (XF) assay for assessing drug-induced mitochondrial effects.
- To establish a robust and sensitive platform for early-stage drug development screening.
Main Methods:
- Utilized galactose-conditioned HepG2 cells exposed to test compounds.
- Simultaneously measured real-time oxygen consumption rate and extracellular acidification rate using a Seahorse XF-96 analyzer.
- Validated the assay with known mitochondrial modulators and automated data analysis with a spline curve fitting model.
Main Results:
- The acute XF assay demonstrated sensitivity in detecting drug-induced changes in mitochondrial activity.
- Validated performance using established drugs affecting mitochondrial function.
- Automated data analysis facilitated efficient processing of screening data.
Conclusions:
- The acute extracellular flux (XF) assay is a robust and sensitive screening platform.
- This method effectively evaluates drug-induced effects on mitochondrial activity in whole cells.
- The assay supports earlier identification of mitochondrial liabilities in drug development.
