Isolation of Murine Endothelial Cells

Mice lung endothelial cells were isolated as described by Xu et al. [36 (link)], with minor modifications. Briefly, lungs were minced, digested with 0.3% collagenase type IV (Invitrogen), and sieved through a 70 μm pore cell strainer (BD Falcon, Atlanta, GA). Cells were then centrifuged for 5 min, 500 rpm at 4 °C. 1 × 10⁶ cells were incubated with 1:100 dilution of Fc Block (BD Pharmingen, San Diego, CA) added to the cells for 30 min at 4 °C. Cells were then stained with PE/Cy7 conjugated anti-CD45 and PE-CF594 conjugated anti-CD31 for 1 h at 4 °C. Post-staining, cells were washed with FACS buffer (PBS supplemented with 0.5% BSA). CD45^–CD31⁺ ECs were sorted with BD Influx Cell Sorter (BD Biosciences).
For isolation of ECs from mice cerebrum, cells were isolated using the Neural Tissue Dissociation Kit (P) and Myelin Removal Beads II (Miltenyi Biotec, Bergisch Gladbach, GE), as described previously [37 (link), 38 (link)]. Cells were first blocked with Mouse BD Fc Block (clone 2.4G2, BD Pharmingen) on ice for 30 min and then stained for 1 h at 4 °C with gentle rotation with a combination of fluorophore-conjugated anti-mouse Abs as described above for isolation of ECs from mice lung tissue.