Brain MVEC (bMVEC) were isolated by incorporating and slightly modifying previously described methods [35 (link),36 (link),37 (link),38 (link)]. Brains of 6–8 wk old male C57BL6/J mice (4–6 animals per isolate), were excised and stored in serum-free DMEM (Sigma, St. Louis, MO, USA) on ice before surgical removal of the olfactory bulbs, cerebellum and mid-brain white matter. The remaining cortical tissue was rolled on sterile filter paper and subsequently digested in DMEM containing 2 mg/mL collagenase A (Roche, Basel, Switzerland) and 10 µg/mL DNase I (Roche) at 37 °C for 1 h, with gentle rotation. Digested tissue was pelleted at 290× g and resuspended in DMEM containing 20% BSA (Sigma) (w/v), then myelin fraction was separated by centrifugation at 1000× g for 10 min. The cell pellet was resuspended and filtered through a 70 µM nylon mesh and collected following centrifugation at 290× g. Cells were further digested in 2 mg/mL collagenase/dispase (Roche) and 10 µg/mL DNase I at 37 °C for 30 min. Following one wash in DMEM at 290× g, bMVEC were selected in medium supplemented with 4 µg/mL puromycin dihydrochloride (Sigma) [39 (link),40 (link)] for the first 4 days.
Free full text: Click here
