The acute extracellular flux (XF) assay to assess compound effects on mitochondrial function.
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.
GlaxoSmithKline, Research Triangle Park, NC, USA ruolan.h.wang@gsk.com.
Ruolan
R
Wang
GlaxoSmithKline, Research Triangle Park, NC, USA.
Steven J
SJ
Novick
GlaxoSmithKline, Research Triangle Park, NC, USA.
James B
JB
Mangum
GlaxoSmithKline, Research Triangle Park, NC, USA.
Kennedy
K
Queen
Seahorse Bioscience, North Billerica, MA, USA.
David A
DA
Ferrick
Seahorse Bioscience, North Billerica, MA, USA.
George W
GW
Rogers
GlaxoSmithKline, Research Triangle Park, NC, USA.
Julie B
JB
Stimmel
D001331:Automation; D004305:Dose-Response Relationship, Drug; D004353:Drug Evaluation, Preclinical / Q000379:methods*; D056945:Hep G2 Cells; D057166:High-Throughput Screening Assays; D006801:Humans; D008928:Mitochondria / Q000187:drug effects* / Q000378:metabolism*; D015203:Reproducibility of Results; D054852:Small Molecule Libraries
D054852:Small Molecule Libraries
acute; mitochondrial dysfunction; screening assay; toxicities