Endothelial cell permeability assay

To assess the cytoprotective effects of APC, the cell permeability assay was performed using a previously described method [33]. Briefly, HUVECs were plated in the upper chamber of the Transwell system until confluence to a monolayer. They were subsequently incubated with fresh culture medium containing different concentrations of APC for 3 h, followed by LPS treatment (100 ng/mL) for 4 h to induce permeability. After washing with PBS, the medium in the upper chamber was replaced with Evans blue (0.67 mg/mL) diluted in the growth medium containing 4% bovine serum albumin. The medium in the lower chamber was replaced with fresh growth medium. Ten min later, the absorbance of the dye was detected at 650 nm in a 50 μL sample (1:3 dilution) obtained from the lower chamber.