MTT assay

HT22 cell viability was assessed using the MTT assay. In brief, cells were seeded onto 96-well plates and cultured until 70% confluency. The cells were pre-incubated with the final concentrations of GAS (200 μM) for 1 h and then exposed to CoCl₂ (200 μM) for 24 h. After discarding the medium, 200 μL of 0.5 mg/mL MTT solution was added to each well and incubated for 3 h. Then, the supernatant was removed and 150 μL of dimethylsulfoxide was added, followed by thorough mixing. Optical density (OD) values were measured at 490 nm using a microplate reader (Thermo Fisher Scientific, Waltham, MA, USA). The cell survival rate (%) was calculated using the following equation:

cell viability inhibition(%) = [(OD_Control − OD_Treated)/OD_Control)] × 100.