MTT assay

An MTT assay was used to estimate cell viability. Briefly, HUVECs were seeded into 96-well plates (BD Falcon, USA), at a density of 2 × 10³ cells/well in DMEM supplemented with 10 % FBS. One day after plating, the cells were washed and incubated in serum-free medium for 12 h. The cells were then washed again. To explore the cytotoxicity of BYHWD on HUVECs, cells were incubated with various concentrations of BYHWD (5–45 mg/ml) for 24 h. To explore the effects of BYHWD on H₂O₂-induced cytotoxicity, cells were pretreated for 6 h with culture medium containing different concentrations of BYHWD (5, 15, and 30 mg/ml). The cells were then exposed to H₂O₂ (320 μmol/l) diluted in culture medium for 6 h. After H₂O₂ exposure, the culture supernatant was removed and cells were washed with PBS before incubating in 150 μl MTT solution (5 mg/ml) in culture medium at 37 °C for 3 h. After removal of the MTT solution, the colored formazan crystals were dissolved in 100 μl DMSO. Plates were gently shaken for 10 min before measuring the optical density at 490 nm using an enzyme-labeled instrument (Biotek ELX 800/FLX800).