Isolation of Capillary Endothelial Cells

Individual capillary endothelial cells were isolated as previously described (Longden et al., 2017 (link)). Brains were denuded of surface vessels with an aCSF-wetted cotton swab, and two 1-mm-thick brain slices were excised and homogenized in ice-cold aCSF using a Dounce homogenizer. The brain homogenate was filtered through a 70 µM filter, and capillary networks that were captured on the filter were transferred to a new tube. Individual capillary endothelial cells were isolated by enzymatic digestion with 0.5 mg/ml neutral protease (Worthington Biochemical Corporation, USA) and 0.5 mg/ml elastase (Worthington Biochemical Corporation) in endothelial cell isolation solution composed of 5.6 mM KCl, 55 mM NaCl, 80 mM sodium glutamate, 2 mM MgCl₂, 0.1 mM CaCl₂, 4 mM glucose, and 10 mM HEPES (pH 7.3; all salts from Sigma-Aldrich, Inc) for 45 min at 37°C. After the first digestion, 0.5 mg/ml collagenase type I (Worthington Biochemical Corporation) was added, and a second 2-min incubation at 37°C was performed. Digested networks were washed in ice-cold endothelial cell isolation solution, then triturated with a fire-polished glass Pasteur pipette to produce individual endothelial cells.