MTT Assay for Cell Viability

Cell metabolic activity and cell viability were determined using a Thiazolyl Blue Tetrazolium Bromide (MTT) (M5655, Sigma, Poole, UK) assay, as described previously [51 (link)]. SHSY-5Y cells were seeded at 3 × 10⁴ cells/well in 96-well plates with growth medium (10% FBS). After 24 h, undifferentiated cells were exposed to ethanol (0–200 mM) diluted in growth media (10% FBS). Differentiated cells were prepared as described above and then treated with ethanol (0–200 mM) diluted in differentiation medium supplemented with 20 ng/mL BDNF. After incubation, spent medium was removed and then replaced with medium containing 10% 5 mg/mL MTT and incubated for 4 h. Plate wells which only received 10% MTT and respective growth medium served as background controls. The generated formazan crystals were suspended in a 1:1 dimethyl sulphoxide (DMSO, D8418, Sigma, Poole, UK)–isopropanol (279544, Sigma, Poole, UK) solution. The absorbance of wells was then read at 570 nm using a spectrophotometer (Multiskan Spectrum, Thermo Electron Corporation, Waltham, MA, USA). An average value was calculated from experiments performed in triplicate after the subtraction of blank (negative control) values. Cell viability was expressed as a percentage of survival compared with that from mock-treated cells. The inhibitor concentrations producing 50% loss of viability of cells (IC₅₀ values) were obtained from the concentration–response curves and expressed as mean ± standard deviation (SD).