Immune Cell Profiling of Spleen and Skin

Single‐cell suspension of freshly isolated splenocytes and total skin was prepared as described earlier (Novoszel et al, 2021 (link)) and subsequently blocked with anti‐CD16/32 antibody (BioLegend). Samples were stained with following fluorophore‐conjugated primary antibodies (Biolegend) for 30 min at 4°C: Ly6G, CD11b, CD11c, CD19, F4/80, MHC class II, Ly6C, and CD3ε (spleen) and B220, BST‐2, CD3ε, CD11c, CD11b, CD19, F4/80, Ly6C, Ly6G, MHC class II, TCR γδ, and XCR1 (skin, two panels; see Appendix Table S2). After incubation, cells were washed, filtered, and stained with SYTOXblue viability dye (Thermo Fisher) according to the manufacturer's recommendation to exclude dead cells. Flow cytometry was performed on a Fortessa (BD Bioscience) and analyzed by FlowJo v10 software.