Isolation and Identification of Brain Endothelial Cells

Cell fractions obtained following MACS were processed as previously described^{8 (link)}. Briefly, original, CD45−, CD45+, CD45− CD31−, and CD45− CD31+ cell fraction aliquots were incubated with anti-CD16/32 (BioLegend, 14-0161-82) to block Fc receptors. Cells were then labeled with CD45 APC (BioLegend, 103111) and CD31 PE-CF594 (BD Biosciences, 653616). A viability dye (LIVE/DEAD fixable green, Molecular Probes, L34969) was added to the previous panels to discriminate live cells. Endothelial cells from mouse brain PFC punches were identified as CD45− and CD31+ cells. All analyses were performed on BD LSR II and data were analyzed with FACS Diva software (BD Biosciences, v.6.1.3).