2.9. Cell viability MTT assay

Cells were seeded in 96-well plates at a density of 2.5 × 10³ cells per well and treated 24 h later with the indicated concentrations of TMZ, rapamycin, and cyclopamine (Sigma) for 72 h in sextuplicates. Then, cells were incubated with MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) 0.25 mg/ml (Sigma) for 3 h. Formazan produced by viable cells was dissolved in 150 μl of DMSO and absorbance determined at 570 nm in a microplate reader (Multiskan Ascent Thermo Electron Corporation, Waltham, MA, USA).