Cell Viability Assay for Endothelial Cells

The cell viability assay was carried out as described [46 (link)] using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT, Merck) with minor modifications. BAECs were seeded at a density of 1.0 × 10⁴ per well in a 96-well culture plate (four replicates for each treatment), and incubated with ZEN at various concentrations (0, 10, 30 or 60 μM) for 24 h or with 30 μM ZEN for various time points (4, 8, 16 or 24 h). After the ZEN treatments, the cells were incubated with 5 mg/mL MTT and further incubated for 1 h at 37 °C. The cells were then treated with dimethylsulfoxide (DMSO) for 10 min, and the absorbance was read at 570 nm using a 96-well microtiter plate reader (BioTek Instruments, Winooski, VT, USA).