Evaluating Cell Viability via Caspase and MTT Assays

Cell viability was detected by determining caspase-3 and -9 activity, as well as by MMT assay. Caspase-9 and -3 activities were determined using caspase-assay kits (Beyotime Institute of Biotechnology), which detect the production of the chromophore p-nitroanilide after its cleavage from the peptide substrate DEVD-p-nitroanilide and LEHD-p-nitroanilide (31 (link)). MMT assay was conducted as previously described (26 (link)). Briefly, the cells were seeded in a 96-well plate at a density of 1×10⁶ cells in triplicate for 7 days at 37°C with 5% CO₂. Subsequently, 20 μl 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) (5 mg/ml; pH 7.4; Sigma-Aldrich) were added to the cells for 4 h. The supernatants were then discarded and 100 μl dimethyl sulfoxide (Sigma-Aldrich) was added to each well for 10 min. The OD of the samples was measured at an absorbance of 490 nm using a spectrophotometer (Epoch 2; BioTek Instruments, Inc., Winooski, VT, USA). The assay was repeated 3 times.