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J Biomol Screen. 2015 Mar;20(3):422-9. doi: 10.1177/1087057114557621. Epub 2014 Nov 7.

The acute extracellular flux (XF) assay to assess compound effects on mitochondrial function.

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GlaxoSmithKline, Research Triangle Park, NC, USA
GlaxoSmithKline, Research Triangle Park, NC, USA.
Seahorse Bioscience, North Billerica, MA, USA.


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.


acute; mitochondrial dysfunction; screening assay; toxicities

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