ApoTox-Glo™ Triplex Assay Technical Manual

Literature # TM322

The ApoTox-Glo™ Triplex Assay combines three Promega assay chemistries to assess viability, cytotoxicity and caspase activation events within a single assay well. The first part of the assay simultaneously measures two protease activities; one is a marker of cell viability, and the other is a marker of cytotoxicity. The live-cell protease activity is restricted to intact viable cells and is measured using a fluorogenic, cell-permeant, peptide substrate (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. A second, fluorogenic cell-impermeant peptide substrate (bis-AAF-R110) is used to measure dead-cell protease activity, which is released from cells that have lost membrane integrity. Because bis-AAF-R110 is not cell-permeant, essentially no signal from this substrate is generated by intact, viable cells. The live- and dead-cell proteases produce different products, AFC and R110, which have different excitation and emission spectra, allowing them to be detected simultaneously.

The second part of the assay uses 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.