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Abstract for Homogeneous Luminescent Caspase Assays to Detect
Apoptosis & In Vitro Toxicity
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
We measure protease activity. 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. |
