Homogeneous Luminescent Caspase Assays to Detect Apoptosis and in Vitro Toxicity...

Abstract

Terry Riss, Martha O’Brien, Andrew Niles, Rich Moravec, Marni Amburn, Deborah Bishop, Mary Sobol, Kay Rashka, Bill Daily* and Mike Scurria*
Promega Corporation, 2800 Woods Hollow Road, Madison WI 53711 and *Promega Biosciences, 277 Granada Drive, San Luis Obispo, CA 93401

Luminogenic substrates have been developed for the measurement of caspase-3/7, -8, & -9 activities as in vitro markers of apoptosis as well as for screening for caspase inhibitors. The homogeneous format of the Caspase-Glo™ Assays was enabled by utilizing a mutant form of beetle luciferase that is stable in the conditions necessary to lyse cells and maintain caspase activity for hours. The Caspase-Glo™ Assay procedure is to add reagent directly to cells in multiwell plates, incubate for 30 min-1 h and record luminescence. There is a linear relationship between the luminescent signal and caspase activity. The luminescent format is 50-fold more sensitive than fluorescent assays for detection of recombinant caspase-3 and can detect as few as 20 apoptotic cells. The stability of the reagents and glow-type luminescent signal are compatible with automation for HTS. Multiplexing of luminescent and fluorescent caspase assays is possible.