Viability of H9c2 cells determined by MTT assay

H9c2 cells in the logarithmic growth phase were collected and seeded into 96-well plates. After 24 h incubation, the cells were divided into the control, model and sevoflurane groups. The cells in the control group received no treatment, cells in the model group were treated with 400 µM H₂O₂, and cells in the sevoflurane group were pretreated with sevoflurane followed by treatment with 400 µM H₂O₂. The sevoflurane pretreatment was performed as previously mentioned (11). H9c2 cells were placed in a sterile sealed container connected with the breathing circuit of an anesthesia machine. The sevoflurane vaporizer was filled with sevoflurane and the vaporizer dial was set at 2.5%. The concentration of sevoflurane in the outlet of the closed container was monitored. When it reached 2.5%, the sevoflurane flow was maintained for 20 min, after which the cells were returned to a normal incubator for another 10 min (11). The hydrogen peroxide treatment was carried out by incubation of the cells with a final concentration of 400 µM H₂O₂ to generate the H₂O₂ stress injury model (12). After 4 h incubation with H₂O₂, the culture medium was discarded, and the wells were washed three times with phosphate-buffered saline (PBS), followed by the addition of 100 µl MTT solution (5 mg/ml) per well and incubation for 4 h. Then, 100 µl dimethyl sulfoxide (DMSO) was added to each well, followed by agitation for 10 min and measurement of absorbance value at 570 nm using a microplate reader (model 680; Bio-Rad Laboratories, Hercules, CA, USA). The cell survival rate was calculated using the formula: Survival rate (%) = 100 × (OD value of experimental group/OD value of control group).