Manual macs

Single-cell suspensions of indicated tissues were labeled with antibodies from eBioscience, Biolegend, Tonbo or R&D Systems. Clones: CD4 (RM4-5), CD8α (53-6.7), CD103 (M290), TCRβ (H57-597), CD44 (IM7), H-2K^b (AF6-88.5), CD5 (53-7.3), CD25 (PC61), PD-1 (J43), TCRγδ (GL3), CD122 (TM-b1), α₄β₇ (DATK32), NK1.1 (PK136), T-bet (4B10), V_α2 (B20.1), V_α3.2 (RR3-16), V_β2 (B20.6), V_β3 (KJ25), V_β4 (KT4), V_β5.1/5.2 (MR9-4), V_β6 (PR4-7), V_β9 (MR10-2), Ki-67 (16A8), CCR4 (2G12), CCR7 (4B12), CCR9 (CW-1.2), CXCR3 (CXCR3-173), CXCR4 (L276F12), CD69 (H1.2F3), Qa2 (69HI-9-9), CD45.1 (A20), CD45.2 (104). Biotinylated CD1d/PBS57 monomers were obtained from US National Institutes of Health tetramer facility and incubated with PE, APC or PE/Cy7 Streptavidin to fluorochrome-labeled multimers. Cells were stained for 20 min at 20 °C. When staining for CCR4 or CCR7, the cells were incubated for 30 min at 37 °C. For S1PR1 staining thymic samples were enriched for IELp by depletion of CD4⁺ cells (Miltenyi, manual MACS). Enriched cells were labeled with 10 μg Rat IgG2 anti-mS1PR1 (R&D Systems, clone 713412) per 1 × 10^{6 (link)} cells for 30 min at 4 °C. The samples were then labeled with biotin anti-rat IgG (clone G28-5) for 20 min on ice and subsequently incubated with streaptavidin-fluorochrome. Before staining with other markers, the samples were blocked with 5% rat serum and 1% free streptavidin.