Aortic endothelial cell isolation

Mouse aortic endothelial cells were isolated following previously described methods (Kobayashi et al., 2005), with modifications. Briefly, mice were perfused via the left ventricle with PBS, the aorta was isolated and filled with 2% collagenase II (Worthington) in serum-free DMEM, and then closed at the ends with 7–0 suture. The vessel was digested for 30–45 min in 10% FBS at 37C, and endothelial cells were flushed out into EC culture medium, and onto a collagen I-coated plate. EC culture medium consisted of DMEM with 10% FBS and 10 mg/mL endothelial growth supplement (ECGS, Biomedical Technologies) with primocin (InvivoGen). Typical isolations of ~1000 cells expanded to ~10,000–50,000 cells. Cells were then FACs-sorted (BD Aria) on endothelial markers, eGFP for Cdh5(PAC)-CreERT2; Rosa26-mTmG on Pecam+ and Icam2+ for Cdh5(PAC)-CreERT2; Rosa26-mTmG; Rbfox2^lox/lox mice and littermate controls.