Cytotoxicity assay in vitro

According to procedures described previously^31,36,37, the cytotoxicity of doxorubicin (DOX, Dalian Meilun Bio, China) was investigated by MTT assay. Briefly, cells were plated into 96-well plates at a density of 1 × 10⁴ cells/well, and cultured at 37°C in a humidified atmosphere with 5% CO₂. After overnight culture, the cells were treated with different concentrations of DOX for 24 h. The subsequent procedures were the same as the above cell proliferation assay. Culture medium was used as a blank control, and the absorbance of untreated cells was considered 100% viability. Cell viability was computed according to the following equation: Cell viability = (OD_treated/OD_control) × 100%. Each sample was assayed in triplicate in three independent experiments.