Caspase 3 fluorescent assay kits

Caspase-3 and caspase-9 activities were detected by Caspase-3 Fluorescent Assay Kits and Caspase-9 Fluorescence assay kits (both from Clontech). The above treated cells were harvested and lysed with cell lysis buffer for 10 min. After centrifuge, 50 μl of 2 Reaction Buffer/DTT Mix and 5 μl specific substrate of DEVD-AFC (for caspase-3) or LEHD-AMC (for caspase 9) were added to the supernatants for further incubation at 37°C. One hour later, caspase activities were detected by a fluorometer (model FL600, Bio-Tek Instruments).

The activities of caspase-3 and caspase-9 were evaluated using Caspase-3 Fluorescent Assay Kits and Caspase-9 Fluorescence Assay Kits (Clontech, Palo Alto, CA), respectively, according to the company's specifications. Briefly, cells under various conditions were lysed with lysis buffer and then centrifuged at 4°C to prepare the supernatants. Then, the reaction buffer containing 5 μl of the specific substrate of DEVD-AFC for caspase-3 or LEHD-AMC for caspase-9 was supplemented into supernatants and incubated for 1 h at 37°C. All specimens were subsequently analyzed using an FL600 fluorescent plate reader (BioTek Instruments, VT, USA) to determine the fluorescent intensity.