The Mitochondrial ToxGlo™ Assay is a cell-based assay method that employs a sequential addition, multiplexed assay chemistry for predicting potential mitochondrial dysfunction as a result of xenobiotic exposure. The assay is based on the differential measurement of biomarkers associated with changes in cell membrane integrity and cellular ATP levels relative to vehicle-treated control cells during short exposure periods. Cell membrane integrity is first assessed by measuring the presence or abscence of a distinct protease activity associated with necrosis using a fluorogenic peptide substrate (bis-AAF-R110) to measure "dead cell protease activity". The bis-AAF-R110 Substrate cannot cross the intact membrane of live cells and therefore gives no signal with viable cells. Next, ATP is measured by adding an ATP detection reagent, resulting in cell lysis and generation of a luminescent signal proportional to the amount of ATP present. The two sets of data can be combined to produce profiles representative of mitochondrial dysfunction or non-mitochondrial related cytotoxic mechanisms.
Mammalian cells generate ATP by mitochondrial (oxidative phosphorylation) and non-mitochondrial (glycolysis) methods. To achieve optimal mitochondrial responsiveness, it may be necessary to refine cell culture conditions. Replacing glucose-supplemented medium with galactose-containing medium may increase cellular oxygen consumption and augment mitochondrial susceptibility to mitotoxicants.
Features - Benefits
Distinguish Primary Mitochondrial Dysfunction from Secondary Cytotoxic Events: Cell-based, multiplexed method measures ATP (a proximal measure of mitochondrial function) in conjunction with a membrane integrity biomarker to distinguish primary mitochondrial dysfunction from secondary cytotoxic events directly in the same sample well.
Predictive for Mitochondrial Toxicities: Produces profiles that are consistent with mitochondrial toxicity and discernible from other non-mitotoxic mechanisms of cell death.
Easy to Implement: The assay uses a simple sequential “add-mix-read” format.
Fast: Quickly assess potential mitochondrial liabilities in under an hour.
Cost-Effective: Assays are performed directly in cell culture plates using standard multimode detection instrumentation.
Flexible and Easily Automated: The volume of reagent addition can be scaled to meet throughput needs; the assay is amenable to automation in 96- and 384-well plates.
Applications
Profile chemical libraries or medicinal modifications for mitochondrial toxicity liability.
References
Niles, A.L. et al. (2007) A homogeneous assay to measure live and dead cells in the same sample by detecting different protease biomarkers. Anal Biochem.366, 197-206.
Marroquin, L.D. et al. (2007) Circumventing the Crabtree effect: Replacing media glucose with galactose increases susceptibility of HepG2 cells to mitochondrial toxicants. Toxicol. Sci97, 539-47.
Rossignol, R. et al. (2004) Energy substrate modulates mitochondrial structure and oxidative capacity in cancer cells. Cancer Res.64, 985-93.
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