Isolation of Mouse Lung Endothelial Cells

Mouse lung ECs were isolated as previously described^{23 (link)}. Klf2^+/− mice (B6; 129S4-Klf2^tm1.1Hhn/J, Stock# 026926) were obtained from The Jackson Laboratory. Briefly, lungs from three Klf2^+/− or three Klf2^+/+ mice were minced into pieces. The lung pieces were digested using pre-warmed (37 °C) type I collagenase (#9001-12-1, Gibco) in a cell incubator at 37 °C for 45 min. The cell suspension was then filtered and centrifuged at 1200 rpm. The precipitates were resuspended in PBS containing BSA and penicillin/streptomycin and added CD31 (BD Pharmingen™, #553370) coated Dynabeads™ (Invitrogen) to incubate on a rotor at room temperature for 15 min. The cells in EP tubes were put on Magnetic Separation Rack and washed with growth medium (DMEM + 20%FBS + penicillin/streptomycin + heparin) for 4 times. Cells were resuspended in growth medium and grown in 0.1% gelatin coated 6-well plate. When the cells approached confluence, CD102 (BD Pharmingen™, #553326) coated Dynabeads™ sorting were performed. Then, the isolated cells were used to do the experiments. All animal procedures conformed to the Guideline for the Care and Use of Laboratory Animals published by the U.S. National Institutes of Health and were approved by the Institutional Animal Care and Use Committee of the University of Rochester Medical Center.