The ApoLive-Glo Multiplex Assay measures both the number of viable cells as a marker of cytotoxicity and caspase activation as a marker of apoptosis within a single assay well to determine the mechanism of cell death. The first part of the assay measures the activity of a protease marker of cell viability. The live-cell protease activity is restricted to intact viable cells and is measured using a fluorogenic, cell-permeant, peptide substrate (glycyl-phenylalanyl-amino fluorocoumarin; GF-AFC). The substrate enters intact cells, where it is cleaved by the live-cell protease activity to generate a fluorescent signal proportional to the number of living cells. This live-cell protease becomes inactive upon loss of cell membrane integrity and leakage into the surrounding culture medium. The second part of the assay uses the Caspase-Glo Assay technology to detect caspase activation, which is a key biomarker of apoptosis. The Caspase-Glo Assay provides a luminogenic caspase-3/7 substrate, which contains the tetrapeptide sequence DEVD, in a reagent optimized for caspase activity, luciferase activity and cell lysis. Adding the Caspase-Glo 3/7 Reagent in an 'add-mix-measure' format results in cell lysis, followed by caspase cleavage of the substrate and generation of a 'glow-type' luminescent signal produced by luciferase. Luminescence is proportional to the amount of caspase activity present.
