Lung endothelial cell isolation and model validation

Mouse lungs were harvested, rinsed and incubated in Dulbecco's modified Eagle's medium containing 2 mg/mL collagenase I for 45 min at 37 °C. 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, in PBS), and incubated with anti-rat immunoglobulin G–coated magnetic beads precoupled with rat anti-mouse PECAM-1 antibody for 30 min at 4 °C in an overhead shaker. Beads were separated from the solution with a magnetic particle concentrator (Dynal MPC-S). The supernatant was kept and the beads were washed five times with buffer 1. Cells-to-CT 1-Step TaqMan kit was used for both the supernatant and the purified endothelial cells, before performing Taqman PCR technology for α1AMPK expression quantification. Data were analyzed with the 2 (-Delta Delta C(T)) method⁵⁷, and expressed as fold of controls.