Isolation of Lung and Liver Endothelial Cells

Lungs and liver from Eng^flox/flox pups were surgically removed, rinsed in ice-cold DMEM and endothelial cells were isolated as described [46 (link)]. Briefly, tissues were minced by the use of scissors, digested in DMEM-3 mg.ml⁻¹ Collagenase A (10103586001, Roche, Basel, Switzerland) for 15 min at 37 °C and then filtered through a 70-µm strainer. The cell suspension was centrifuged at 200× g for 5 min and CD45⁺ cells were removed using Dynabeads sheep anti-Rat IgG (11035, Invitrogen, Waltham, MA, USA) coated with rat anti-mouse CD45 antibody (550539, BD Pharmigen, Franklin Lake, NJ, USA). Endothelial cells were sorted using Dynabeads sheep anti-Rat IgG (11035, Invitrogen, Waltham, MA, USA) coated with rat anti-mouse PECAM1 antibody (550274, BD Pharmigen, Franklin Lake, NJ, USA) according to the manufacturer’s instructions. After washing 5 times with DMEM-0.1% BSA, cells were seeded in 6-well plates. Lung and liver ECs were maintained for two-three passages in Endothelial Cell Growth Medium 2 (C-22011, PromoCell, Heidelberg, Germany) complemented with Fetal Calf Serum, Human Epidermal Growth Factors, Basic Fibroblast Growth Factor, Insulin Like Growth Factor, Human Vascular Endothelial Growth Factor-165, Ascorbic acid, Heparin and Hydrocortisone (C-39211, SupplementPack EC GM2, PromoCell, Heidelberg, Germany).