Determination of reactive oxygen species (ROS) production

The membrane permeable indicator H₂DCF-DA (Invitrogen; Thermo Fisher Scientific, Inc., Waltham, MA, USA) was used to determine ROS production by EPCs. EPCs were loaded with 10 µmol/l H₂DCF-DA in serum-free EGM-2MV medium at 37°C for 30 min and then washed twice with phosphate-buffered saline (PBS). Following treatment with various concentrations of RSV (0.01, 0.1, 1, 5 and 10 µmol/l; Sigma-Aldrich; Merck Millipore, Darmstadt, Germany) for 48 h, the cells were analyzed with a flow cytometer (BD Biosciences, Franklin Lakes, NJ, USA) at an excitation wavelength of 488 nm and an emission wavelength of 525 nm. ROS production was determined by comparing the changes in fluorescence intensity with those of the control. CellQuest Pro software, version 3.1 (BD Biosciences) was used for analysis.

Measurement of NADPH oxidase activity. To evaluate NADPH oxidase activity in EPCs, the lucigenin-derived enhanced chemiluminescence assay (Beyotime Institute of Biotechnology, Shanghai, China) was employed. EPCs were starved in serum-free EGM-2MV medium for 24 h and treated with RSV (0.01, 0.1, 1, 5 and 10 µmol/l), and subsequently washed twice with ice-cold PBS (pH 7.4) and centrifuged at 2,000 × g for 5 min at 4°C. The cells were re-suspended in ice-cold buffer (pH 7.0) containing 1 mmol/l ethylene glycol tetraacetic acid, protease inhibitors, and 150 mmol/l sucrose, and lysed. A Bradford assay was used to determine the total protein concentration, which was adjusted to 1 mg/ml. Every 100 µl of protein sample, including 2.5 µmol/l lucigenin, was measured over 6 min in quadruplicate using NADPH (100 µmol/l) as a substrate in a luminometer counter (Centro LB 960; Berthold Technologies GmbH & Co. KG, Bad Wildbad, Germany).