Genistein Cytotoxicity Assessment via MTT Assay

The cell viability was examined using 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyltetra-zolium bromide (MTT) assay according to the previous method [23 (link)]. In brief, 1 × 10⁴ cells per well were plated in 96-well plates. After 24 h, the cells were treated with the desired concentrations of genistein with or without N-Benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone (z-VAD-fmk, Calbiochem, San Diego, CA, USA), N-acetyl-L-cysteine (NAC, Invitrogen, Waltham, MA, USA) or LY294002 (Cell Signaling Technology, Inc., Danvers, MA, USA). After 48 h, the medium was changed with fresh medium containing 50 μg/mL MTT solution (Invitrogen). After 2 h, the medium was removed and added 100 μL of DMSO. Absorbance at 540 nm was measured using a microplate reader (Molecular Device Co., Sunnyvale, CA, USA). The morphological changes of cells following genistein treatment were observed and visualized by a phase-contrast microscope (Carl Zeiss, Oberkochen, Germany).