TMAO Regulates Adhesion Molecules in HUVECs

Adhesion molecules expression was measured by immunocytochemistry. Briefly, HUVECs were plated in 96-well plates. Confluent HUVECs were serum starved for 6 h and treated with TMAO (0, 10, 50, and 100 μmol/l) for 6 h at 37°C, TNF-α was served as the positive control. Then, HUVECs were incubated with rabbit anti-ICAM-1 (PB0053, Boster) or rabbit anti-VCAM-1 (BA0406, Boster) or rabbit anti-E-selectin (BA0615, Boster) (1:200; Boster, China) in First Antibody Dilution Buffer for 2 h at 37°C. Omission of primary antibody was conducted in negative controls. And then the cells were incubated with a horseradish peroxidase-conjugated antibody (1:1000, Abcam, U.S.A.) for 1.5 h at 37°C. Quantification was performed by measuring the absorbance at 450 nm by a TMB peroxidase EIA substrate kit (Bio-Med Innovation, China) [25 (link)]. PKC inhibitor, staurosporine (2.5 nmol/l), was used at the beginning of the treatment.