2.7. Caspase-3 Activity

The HepG2 cells were incubated with various concentrations of PHMG-P for 16 h or 24 h. Caspase-3 activity was then determined by fluorometrically measuring the substrate cleavage. To evaluate caspase-3 activity, the cell lysates were prepared after their respective treatment with PHMG-P. The assays were performed in 96-well microtiter plates by incubating 50 μL of the cell lysate in 100 μL of reaction buffer (0.5 M HEPES, 1 M NaCl, 1 M DTT) containing the caspase-3 substrate (Ac-DEVD-AMC) at 5 μM. The lysates were incubated at 37 °C for 4 h. Thereafter, the fluorescence was measured at 460 nm, after the excitation at 380 nm, using a Glomax microplate reader (Promega).