Mitochondrial cell-based assay

Promega Corporation has launched of the Mitochondrial ToxGloTM Assay, a cell-based assay that enables researchers to quickly profile large compound libraries or medicinal modifications for their effects on mitochondria by allowing them to distinguish primary mitochondrial dysfunction from secondary cytotoxic events in a single well via a sequential-addition, multiplexed assay chemistry.

The Mitochondrial ToxGlo Assay is based on the differential measurement of biomarkers associated with changes in cell membrane integrity and cellular ATP levels during short xenobiotic exposure periods. Cell membrane integrity is initially assessed by measuring the presence or absence of a distinct protease activity associated with necrosis, using a fluorogenic peptide substrate to measure 'dead cell protease activity'.

The substrate cannot cross the intact membrane of live cells and therefore gives no signal with viable cells. Next, ATP is measured as an indicator of mitochondrial function by adding an ATP detection reagent to the same well of cells, resulting in cell lysis and the generation of a luminescent signal proportional to the amount of ATP present. The two sets of data are then combined to produce profiles representative of mitochondrial dysfunction or non-mitochondrial related cytotoxic mechanisms.

The Mitochondrial ToxGlo Assay has been designed for ease of use, with a simple, sequential 'add-mix-read' format, which enables researchers to quickly assess potential mitochondrial liabilities in under one hour. The assays are performed directly in cell culture microwell plates and data captured using standard multimode detection instrumentation. The assay is easy to automate and can be scaled to meet throughput needs for 96- and 384-well plate formats.

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