Mitochondrial ToxGlo™ Assay Technical Manual

Literature # TM357

The Mitochondrial ToxGlo™ Assay is a cell-based assay method that employs a sequential addition, multiplexed 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 absence 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 insignificant signal with viable cells, relative to non-viable cells. Next, ATP is measured by adding the ATP Detection Reagent, resulting in cell lysis and generation of a luminescent signal proportional to the amount of ATP present. The ATP Detection Reagent consists of an optimized formulation for ATP detection containing luciferin, ATPase inhibitors and thermostable Ultra-Glo™ luciferase. 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