Endothelial cell proliferation assay in vitro

HUVEC (5x10³/well/100 μl medium) were seeded on a 96-well plate with Endothelial Cell Growth Medium (Cell Applications). After 24 hours of culture, the medium was replaced by 100 μl condition medium from AGS shRNA-control or CRT-knockdown cells and treated with 20 ng/ml of VEGF-A recombinant protein (Genetex). At the indicated times, medium containing 50 μl of 5 mg/ml MTT was added to each well for 3 hours. The cells were then gently washed with PBS, followed by the addition of 100 μL of DMSO to dissolve the formazan crystals for 10 minutes. The absorbance was then read at 570 nm. Each experiment was performed in triplicate.