Cck 8 solution

The effect of OGD/R on cell viability was measured using CCK8. Cells were detached with 0.25% trypsin and centrifuged at 1,000 rpm for 3 min. The cells were resuspended in phosphate-buffered saline and plated in 96-well plates at 5 × 10³ cells/well. After the exposure to 1% O₂ + 5% CO₂ + 94% N₂ under hypoxic conditions for 2 h, 4 h, and 8 h, respectively, the cells were reoxygenated in complete medium for 24 h to obtain OGD/R cells and incubated again with 10 µL CCK8 solution (Abmole Bioscience Inc., Houston, TX, USA) for 2 h. The cell viability was then assessed by recording the OD value of each well using a microplate reader (450 nm, Bio-Rad).

The CCK-8 assay was performed to determine cell proliferation as previously described [27 (link)]. Briefly, fibroblasts from different groups were seeded in a 96-well plate and cultured for 24, 48, 72, and 96 h at 37°C, before the CCK-8 solution (AbMole, China) was added. Then the fibroblasts were cultured for another hour followed by the measure of absorbance using a microplate reader (450 nm; Thermo Fisher Scientific).

After OGD/R, BMVECs transfected with VWF-overexpressing plasmids or not were adjusted to the density of 2 × 10³ cells/well and seeded in a 96-well plate (100 μL/well). Following 24 h of incubation, the cells were subjected to DHA treatment and incubation with 10 μL CCK-8 solution (M4839, Abmole Bioscience, Houston, TX, USA) for 1 h. Thereafter, the optical density value was detected by a microplate reader (Synergy HTX, BioTek, Winooski, VT, USA).