Methyl thiazolyl tetrazolium (MTT) colorimetry

Cells were washed with PBS twice when grown to confluence (80% confluent). Cells were inoculated in a 96-well plate (200 μl per well, 6 repeated wells) in a density of 3 × 10³ ~ 6 × 10³. Cells were incubated at 37°C, 5% CO₂ for 24–72 h, and 20 μl of MTT solution (5 mg/ml, Sigma) were added into each well. After 4 h incubation at 37°C and 5% CO₂, the incubation was terminated, and the culture medium was discarded. DMSO (150 μl) (Sigma) was added to each well, and gently shaken for 10 min to promote crystallization dissolution. Absorbance values (OD) were determined with an enzyme-linked immunosorbent detector at 12 h, 24 h and 48 h, respectively. MTT curve was draw with interval time as the X-axis and the OD value as the Y-axis. The experiment was triplicated.