Cell viability assay

Silymarin-induced changes in breast cancer cell viability were assessed using a MTT assay. MDA-MB-231 and MCF-7 cells were seeded into 96-well plate at a density of 2×10⁴ cells/ml (200 µl/well). Following incubation for 24 h at 37°C (5% CO₂), the cells were treated with silymarin 0, 25, 50, 75, 100, 150 and 200 µg/ml for 24 h. We checked the concentrations that based on the previous experiment in the reference (9,10). Silymarin was dissolved in alcohol and diluted in the media. After treatment, the medium was discarded, and 40 µl MTT solution (5 mg/ml) was added prior to incubation for a further 2 h. Subsequently, 100 µl of DMSO was added to each well to dissolve the formazan crystals and the absorbance was recorded at 595 nm using a microplate reader (Bio-Rad, Laboratories, Inc). Untreated control cells were used as the comparative control.