hNSPs, hNPCs, primary human immature and mature neurons were cultured in 12-well tissue culture plates and treated in triplicate with different concentrations of EtOH (10 mM, 25mM, 50 mM and 75 mM) for 6, 24 and 48 h. After treatments, cells were incubated for 2 h at 37 °C with 150 µl of MTT (3-(4 5-dimethylthiazol-2-yl)-2 5-diphenyltetrazolium bromide) at 0.5 mg/ml working solution. The converted insoluble purple formazan was solubilized with 500 µl of acidic isopropanol (0.004 M HCl in isopropanol). Absorbance of the converted formazan was measured at a wavelength of 570 nm with a background subtraction at 650 nm. hNPCs were plated in 12-well tissue culture plates and transfected with 1 or 2 µg of pcDNA3.1-Mcl-1L or pcDNA3.1-Mcl-1S. Total DNA concentrations were adjusted with empty vector pcDNA3.1 per transfection. 24 h after transfections, cells were treated in absence or presence of EtOH 50 mM for 24 h and then MTT viability assays were performed as described above.
