Primary Mouse Brain Endothelial Cell Isolation

Primary BMECs were isolated from 8-week-old CD-1 mice as previously described [2 (link)]. Briefly, isolated BMECs were cultured using BMEC medium, which contained Dulbecco’s modified Eagle’s medium (DMEM)/F12 supplemented with 20% plasma-derived fetal bovine serum (Animal Technologies), 1% GlutaMAX (Thermo fisher Scientific), basic fibroblast growth factor (bFGF, 1 ng/ml; Roche Life Sciences), heparin (100 μg/ml), insulin (5 μg/ml), transferrin (5 μg/ml), selenium (5 ng/ml) (Thermo fisher Scientific), and gentamicin (50 μg/ml) (Sigma Aldrich). Puromycin (4 μg/ml) (Sigma Aldrich) for the first 48 h after plating. Cells were maintained at 37 °C in a CO₂ incubator for 24 h. Culture medium was changed at 24 h after plating to remove non-adhering cells, RBCs, and debris. At 48 h after plating, the medium was changed to BMEC medium in the absence of Puromycin. The purified primary BMECs were used to construct in vitro models when reached 80% confluency.