Anti human cd31 conjugated to pe

For mouse experiments, cells were co-stained with anti-mouse CD8α conjugated to PE (Tonbo Bioscience), anti-mouse CD4 conjugated to violetFluor450 (Tonbo Bioscience), anti-mouse CD44 conjugated to BV605 (Biolegend), anti-mouse CD62L conjugated to APC (eBioscience), and anti-mouse CD31 conjugated to PE-Cy7 (eBioscience). All stains were completed on ice to prevent internalization. For human studies, cells were co-stained with anti-human CD8α conjugated to APC (eBioscience), anti-human CD4 conjugated to PerCP-Cy5.5 (eBioscience), anti-human CD45RO conjugated to APC-efluor780 (eBioscience), anti-human CD45RA conjugated to evolve605 (eBioscience), and anti-human CD31 conjugated to PE (eBioscience). All stains were completed on ice to prevent internalization. Cells were fixed in 1% paraformaldehyde (Fisher Scientific) before flow cytometric analysis. Data was collected on a FACS LSR Fortessa using FACS Diva software (BD Biosciences). Analysis was performed using Flow Jo software (Tree Star). For mouse and human studies, lymphocytes were gated based on forward and side scatter, and then by CD4⁺ or CD8a⁺. In the mouse, naïve cells were gated based on expression of CD62L⁺CD44⁻ and activated cells were gated by CD44⁺CD62L⁻. In the human, naïve cells were gated by CD45RA⁺CD45RO⁻ expression, while activated cells were gated by CD45RO⁺CD45RA⁻ expression.