MTT assay for neuroprotection

Cell viability was measured using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. PC12 cells (1 × 10⁵ cells·ml⁻¹) cultured in a 96-well plate (BD Biosciences) were pretreated with 1/1,000 dilution of EEB for 10 min, followed by the addition of 200 μM corticosterone (Sigma-Aldrich, St. Louis, MO, USA) for 48 h. After sample treatment, 100 μl of culture medium and 10 μl of MTT (5 mg·ml⁻¹) were added and the cells were incubated for 6 h. The MTT formazan formed was dissolved in 100 μl of 10% SDS (w/v) and the absorbance was measured using a microtiter plate reader (Powerscan HT; Dainippon Sumitomo Pharma Co. Ltd., Osaka, Japan).