Huh-7 Cell Culture and Cytotoxicity Assay

Huh-7 cell line (Korean Cell Line Bank, Seoul, Korea) was cultured in complete RPMI-1640 medium supplemented with 10% fetal bovine serum, 100 IU/mL penicillin, and 100 µg/mL streptomycin. The cells were incubated at 37 °C in a humidified atmosphere containing 5% CO₂. Cytotoxicity of C militaris extracts on Huh-7 cells was evaluated by MTT (3-(4-5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assays. Briefly, cells were plated in 96-well plates at 1 × 10⁴ cells/well. After 24-hour incubation, the cells were treated with various concentrations of the Cordyceps militaris ethyl acetate extract fraction (CMEAF) for 24 hours. At the end of the incubation, 50 µL of the MTT solution (3 mg/mL in phosphate-buffered saline) was added to each well. After 2-hour incubation, the medium containing MTT solution was replaced with isopropanol for extraction of dye. After incubation for 30 minutes, absorbance was measured on VERSAmax microplate reader (Molecular Devices) at 570 nm.