Anti rat immunoglobulin g coated magnetic beads

Lungs from 8-week adult Atg5^lox/lox and Cdh5.Cre-Atg5^lox/lox mice were harvested and incubated in 5 mL Dulbecco’s modified Eagle’s medium containing 2 mg/mL collagenase I (Invitrogen, 17,100–017) for 45 min at 37 °C with shaking every 15 min followed by filtering through a 40-μm nylon mesh (BD Falcon, 352,340). The cells were then centrifuged at 1,000 g for 5 min at 4 °C, re-suspended in buffer 1 (0.1% bovine serum albumin, 2 mM EDTA, pH 7.4, in PBS), and incubated with anti-rat immunoglobulin G-coated magnetic beads (Invitrogen, 1103) pre-coupled with rat anti-mouse platelet/endothelial cell adhesion molecule-1 (PECAM-1; MEC13.3, BD Pharmingen,550,274) for 30 min at 4 °C in an over-head shaker. Beads were separated from the solution with a magnetic particle concentrator (Dynal MPC-S, Invitrogen). The beads were washed five times with buffer 1 and centrifuged for 5 min at 1000 g, and the supernatant was removed. The purified endothelial cells were then cultured in complemented EGM-2 (PromoCell, 22,010). For western blot analysis, lung endothelial cells (2 × 10⁵) were seeded in 60-mm dishes and cultured for 24 h in ECGM-2 at 37 °C and 5% CO_2, followed by 8-h starvation in EBM (PromoCell) before 100 ng/ml VEGF-A treatment when indicated.

We harvested mouse lungs between P15 and P21, minced them and incubated them in 5 mL Dulbecco’s modified Eagle’s medium containing 2 mg/mL collagenase I (Invitrogen) for 45 min at 37 °C with shaking every 15 min followed by filtering through a 40-μm nylon mesh (BD Falcon). The cells were then centrifuged at 1000 × g for 5 min at 4 °C, resuspended in buffer 1 (0.1% bovine serum albumin, 2 mM EDTA, pH 7.4, in PBS), and incubated with anti-rat immunoglobulin G-coated magnetic beads (Invitrogen) precoupled with rat anti-mouse platelet/endothelial cell adhesion molecule-1 (PECAM-1; MEC13.3, BD Pharmingen, 553370) for 30 min at 4 °C in an overhead shaker. Beads were separated from the solution with a magnetic particle concentrator (Dynal MPC-S, Invitrogen). The beads were washed five times with buffer 1 and centrifuged for 5 min at 1000  × g, and the supernatant was removed. The purified endothelial cells were then cultured in ECGM-2 (Promocell). For western-blot analysis, lung endothelial cells (2 × 10⁵) were seeded in 60-mm dishes and cultured for 24 h in ECGM-2 at 37 °C and 5% CO₂.