Flow Cytometry and Cell Sorting Protocol

Flow cytometry was performed using a BD LSRFortessa analyzer (BD Biosciences), whereas cell sorting was performed using an XDP cell sorter (Beckman Coulter), as described (26 (link)). The purity of isolated populations was typically >98%. The following were used (eBioscience unless otherwise stated): anti-CD4 allophycocyanin (FM4-5), anti-CD8 FITC (53-6.7), anti-TCRβ (H57-597), anti-NK1.1 PE/PE Cy7 (PK136), anti-CD24 PerCP Cy5.5 (M1/69), anti-CD44 A700/PE Cy7 (IM7), anti-RORγt PE (AFKJS-9), anti-CD16/32 (93), anti-CD45 allophycocyanin Cy7/allophycocyanin eFluor 780 (30-F11), anti-EpCAM1 allophycocyanin (G8.8), anti-Ly51 PE (6C3), anti–I-A^b biotin (Becton Dickinson, AF6-120.1), anti-Aire 488 (5H12), anti-ICOSL biotin (HK5.3), and anti-CD80 Brilliant Violet 421 (16-10A1; BioLegend). Biotinylated Abs were detected with streptavidin PE Cy7. Brilliant Violet 421/allophycocyanin–conjugated CD1d tetramers loaded with PBS57 were from the National Institutes of Health Tetramer Facility. For RORγt staining, cells were permeabilized with the Foxp3 staining kit (eBioscience). Aire staining (27 (link)) and staining using anti–RANK ligand (RANKL; IK22.5) and anti-CD40L (MR1; BD Biosciences) was performed as described (25 (link)).