Cd31 pe cf594

Original, CD45⁻, CD45⁺, CD45⁻ CD31⁻, and CD45⁻ CD31⁺ cell fraction aliquots obtained following MACS separation 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 NAc 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).

Murine lung endothelial cells (MLECs) were isolated by digestion of the lungs using collagenase I (Worthington, LS 004216) followed by two-step separation of the cell suspension using CD31 MACS beads and ICAM Dynabeads according to instructions [32 (link),33 (link)]. FACS sorting (FACSAria; BD, Heidelberg, Germany) of the MLEC stained with CD31 PE-CF594 (BD, Heidelberg, Germany), CD326 BV421 and CD45 PE-Cy7 (Biolegend, San Diego, CA, USA) and 7-AAD PerCP-Cy 5.5 (ThermoFisher, Frankfurt, Germany) was used for purification as described previously [34 (link)].

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).