MTT Assay for Cell Viability Analysis

For analysis of cell viability, MTT assay was carried out by a procedure similar to that described previously [30] (link). Briefly, cells were seeded in 24-well plate (5×10⁴ cells/well) and grown for 24 h. The cells were then treated with MML at different concentrations (0-80 µM) for 24 h or treated with 60 µM of MML for different time courses (0-24 h). After MML treatment, cells were incubated with medium/MTT (0.5 mg/ml) mixture for 3 h. DMSO was added to dissolve the reduced formazan crystal from MTT and absorbance was read at 540 nm using ELISA plate reader (Bio-Rad, Hercules, CA, USA). To confirm the effect of MML on cell proliferation, live cells were counted by trypan blue staining according to the manufacturer's instructions. In brief, A549 cells were plated in 12-well plate and treated with MML as indicated concentrations for 24 h. After treatment, cells were harvested using trypsin-EDTA (WelGENE Co., Daegu, Korea) and stained with 0.4% trypan blue solution (Gibco, Carlsbad, CA). Living cells were counted by hemocytometer. Cell viability was shown relative to the control.