3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide (MTT) assay

Cells were seeded into 96-well plates at 1 × 10⁴ cell/well and incubated overnight at 37 °C with 5% CO₂. In the medium consisting of 10% FBS, cells were treated with TMZ (KeyGen Biotech Co., Ltd., Nanjing, China), a chemotherapeutic drug of 0, 4, 20, 100, 500, or 2500 μg/mL, for 24 hours respectively. The inhibited concentration of half of the cells (IC50) of TMZ was also detected. Then, 10 μL MTT solution (0.5 mg/mL) was added to each well and incubated for 4 hours. Then, The reaction was terminated by adding 200 μL dimethyl sulfoxide (DMSO) and incubated at 37 °C for 15 minutes after removing the supernatant. The optical density (OD) value at 490 nm was measured by a microplate reader (Bio-Rad, Hercules, CA, USA).