Isolation and Purification of Mouse Lung Endothelial Cells

MLECs were isolated as described previously^{42 (link)}. Briefly, mice were anesthetized, chest cavity was opened, and blood was removed by infusing 10 ml PBS. Lung tissues were diced into ~1 mm³ pieces and cultured in a 60 mm culture dish in growth medium (DMEM containing 2 mM glutamine, 10% FBS, 5% human serum, 50 μg/ml penicillin/streptomycin, 5 μg/ml heparin, and 80 μg/ml EC growth supplement from bovine brain) at 37 °C with 5% CO₂ for 60 h. Then, remove tissue dices from dish and culture the adherent cells for 3 days. Purify the MLEC by biotin-conjugated rat anti-mouse CD31 mAb and BD IMag streptavidin particles plus-DM, and the immunomagnetic separation system (BD Biosciences) following the manufacturer’s instructions. Purified MLECs were characterized by their cobblestone morphology.