Sv40 large t antigen

Lung tissues from three 7–10 day old mice were removed aseptically and rinsed in ice-cold DMEM. After removal of the larger blood vessels, the lungs were minced into ~1×2-mm squares and digested with 1.5mg/ml collagenase A (Roche), at 37°C for 45 minutes on a rotator. The cellular digest was filtered through sterile 70μm nylon mesh and the mesh washed with 15ml 10% FBS-DMEM media to stop digestion. Cells were centrifuged at 400xg for 10 minutes, resuspended in 0.1% BSA/PBS, incubated with anti-CD31-antibody (BD) coated Dynabeads (Invitrogrn), and then separated in a magnetic field. After washing away of unbound cells, remaining cells were seeded on 2% gelatin coated plates cultured with Endothelial cell medium (ScienCell). After 3–5 days, cells were separated again by anti-ICAM2 antibody (BD) coated Dynabeads. The cells were plated at a concentration of 300,000 cells/ml into gelatin-coated T-25 flasks, and subsequently split 1:2 at each passage. After 3 passages, cells were immortalized using lentivirus expressing SV40 large T antigen (Genecopoeia).

Primary lung-derived endothelial cells were isolated from lung tissues of 8 to 10-week-old mice as described [18 (link)]. Briefly, minced lungs were digested with 1.5 mg/ml collagenase A (Roche) and filtered through nylon mesh into 10% FBS–DMEM media. Cells were selected with anti-CD31-antibody (BD Biosciences) and anti-ICAM2 antibody (BD) coated Dynabeads (Invitrogen) and cultured with endothelial cell medium (ScienCell). After 3 passages, cells were immortalized using lentivirus expressing SV40 large T antigen (Genecopoeia).