Tbars assay

Isolation of primary endothelial cells from WT or Dscr-1^−/− mice lung was performed by DynaBeads system (ThermoFisher). Briefly, whole lung tissue from mice was digested by type II collagenase solution containing DNase I, then lysis red blood cells and determined cell number. Cells were incubated with DynaBeads conjugated with rat anti-mouse CD31 antibody (BD Bioscience) and separated into endothelial and non-endothelial fraction by magnetic separating system. After first isolation, cells were cultured in 20%FBS/DMEM containing non-essential amino acid, sodium pyruvate and antibiotics for five days. For second isolation, expanded cells were harvested and incubated with DynaBeads conjugated with rat anti-mouse CD102 antibody (BD Bioscience) to increase endothelial cell purity. Obtained primary endothelial cells from each mouse were cultured until confluent, and incubated in 0.5%FBS/MCDB131 medium for 18 h starvation. 1μM Cyclosporine (CsA) were treated at 30 min before VEGF stimulation. After 24 h stimulation of VEGF (50ng/ml) or PBS as control, cells were collected and performed TBARS assay (Cell Biolabs) to measure malondialdehyde (MDA) according to manufacturer’s instruction. Produced MDA level by oxidative stress were determined at 540nm excitation/590nm emission for fluorometric measurement and quantified concentration of MDA with standard curve.