2.10. MGMT activity assay

MGMT activity was quantified using the MGMT Activity Assay Kit (BioVision, Inc.) according to the manufacturer's instructions. Briefly, 2 × 10⁶ cells were resuspended in 100 μl extract buffer, put on ice for 20 minutes, and centrifuged at 12,000 g for 10 minutes at 4°C. The supernatants were collected and diluted 10 times with ddH₂O, and the protein concentration was measured. The supernatants (containing ~50–200 μg of proteins) were diluted to 85 μl with extraction buffer. Positive and negative control wells were set up, and 10 μl of 10× reaction buffer and 5 μl calpain substrate were added into each well. After incubation in dark at 37°C for 1 hour, the fluorescence intensity of the samples was measured using a plate reader (BioTech) with excitation at 400 nm and emission at 505 nm.