Isolation and Culture of Mouse Lung Endothelial Cells

The lung endothelial cells were isolated from whole lung tissues using CD45 (Miltenyi Biotec, 130-052-301) and CD31 Microbeads (Miltenyi Biotec, 130-097-418) as previously described [22 (link)]. In brief, the harvested lungs were minced and digested with collagenase II (Gibco, 17101015) and filtered through a 100-μm nylon mesh. The cell suspension was resuspended in bovine serum albumin and incubated with anti-CD45-conjugated magnetic beads for negative selection, followed by positive selection with anti-CD31-conjugated magnetic beads. The CD31-positive cells were collected with an MACS separator (Miltenyi Biotec) following the manufacturer’s protocols. The purity of the endothelial cell population was confirmed by immunofluorescence of anti-CD31 (Abcam, ab7388) and anti-VE-cadherin (Affinity, AF6265). The primary mouse lung endothelial cells (MLECs) from 8-week-old male mice were cultured for in vitro experiments. Isolated MLECs were cultured with Dulbecco’s modified Eagle’s medium (DMEM) containing endothelial growth supplement, and the cells between passages 2 and 6 were used for in vitro experiments. The freshly isolated lung endothelial cells from lean and DIO mice were collected immediately to detect the protein levels by western blot.