Measurement of Caspase-3 Activity

Caspase-3 activity was measured as previously described (30 (link)). The activities of caspase-3 were measured using a DEVD-AFC device (BioVision). Cells were collected from monolayers and then lysed in 60 μl of cell lysis buffer for 10 min on ice. After centrifugation, 45 μl of supernatant were extracted and added to a 96-well plate. Next, 50 μl of 2 × reaction buffer (containing 10 Mm dithiothreitol) were added to each sample. Finally, 5 μl of the 1 Mm DEVD-AFC substrate (50 μM final concentration) were added, and the mixture was incubated for 1–2 h at 37°C. The levels of cleaved caspase substrate were measured using a spectrofluorometer (Molecular Devices).