Ab39700

The test cells were plated and treated as described above for the apoptosis and cell cycle studies. The treated cells were harvested by trypsinization at 12 and 24 hr and then tested with caspase-3, -8, and -9 colorimetric assay kits (Catalog No. ab39401, ab39700, and ab65608, respectively; Abcam, Cambridge, UK) according to the manufacturer’s instructions. In brief, the harvested cells were washed with cold PBS and centrifuged at 800 × g. The cell pellet was then lysed by the addition of 50 μL of chilled cell lysis buffer and incubation on ice for 10 min. The lysed mixture was clarified by centrifugation at 10,000 × g for 1 min and the supernatant was transferred to a new microcentrifuge tube. The protein concentration in the supernatant of each sample was measured using the Bradford assay and then adjusted to 100 μg of protein per 50 μL of cell lysis buffer for subsequent application to each well of a 96-well plate. Next, 50 μL of 2× reaction buffer containing dithiothreitol at a final concentration of 10 mM was added into each sample well. After mixing, the respective substrate of each caspase was added to each well and the plate was incubated at 37°C for 1–2 hr. Finally, the absorbance of each reaction at 400–405 nm was measured on a microplate reader. Each experiment was performed in triplicate.