Cytotoxic Effect of DBDS Assessed by MTT Assay

The MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) colorimetric method was used to determine cytotoxicity of DBDS. The logarithmic phase cells were seeded in 96-well plates with 1 × 10⁵ cells/well (volume of the well is 100 μL). Under a humidified atmosphere (5% CO₂, 37 °C), the cells were cultured for 24 h following treatment with vehicle and DBDS (12.5, 25, 50, 100, and 200 µmol/L). After 24 h, 20 μL of MTT solution was added to each well and kept under the same environment for 4 h again. Subsequently, 150 μL of DMSO was added to each well after removing the culture supernatant, vibrated and kept under a darkroom at room temperature for 15 min. The optical density (OD) of the solution was measured with a microplate reader at 490 nm. The survival rate was calculated using the formula, survival rate (%) = OD of the sample-treated group/OD of the control group × 100%.