Plzf 9e12

Single-cell suspensions were prepared from the indicated tissues and stained with fluorescence-conjugated antibodies as previously described (26 (link)). The data were acquired using LSR Fortessa or LSRII flow cytometers (BD Biosciences) and were analyzed using software platforms developed by the EIB Flow Cytometry Facility, CCR, NCI, NIH. Live cells were gated by forward scatter exclusion of dead cells stained with propidium iodide. The following antibodies were used for staining: HSA (M1/69), T-bet (4B10), Foxp3 (FJK-16s), RORγt (AKFJS-9) and isotype control antibodies, all from eBioscience; CD4 (GK1.5 and RM4.5), CD8α (53–6–7), CD69 (H1.2F3), TCRβ (H57-597) and IL2Rβ (TM-β1) from BD Biosciences; CD44 (IM7), NK1.1 (PK136), IL2Rα (PC61), CCR7 (4B12) and PLZF (9E12) from BioLegend. Fluorochrome-conjugated CD1d tetramers loaded with PBS-57 (CD1dTet) and unloaded controls were obtained from the NIH tetramer facility (Emory University, Atlanta, GA). Intracellular Foxp3, PLZF, RORγt, and T-bet proteins were detected using a Foxp3 staining kit according to the manufacturer’s instructions (eBioscience Thermo Fisher).

Fluorescence-conjugated antibodies with the following specificities were used to detect antigens by flow cytometry: CD4 (GK1.5), CD8α (53-6-7), IL-4Rα (M1), CD44 (IM7), γδT cell receptor (GL3), γc (4G3), RORγt (Q31-378), Runx3 (R3-5G4), pSTAT1 (pY701; 4a), and isotype control antibodies, all from BD Biosciences; CD24 (M1/69), IL-2Rβ (TM-β1), IL-7Rα (A7R34), IFNGR1 (2E2), Eomes (Dan11mag), IL-4 (11B11), IL-17 (eBio17B7) and T-bet (4B10) from Invitrogen; CXCR3 (CXCR3-173), TCRβ (H57-597), IFNγ (XMG1.2), Ikaros (2A9), and PLZF (9E12) from Biolegend. CD1d tetramers loaded with PBS-57 were obtained from the NIH tetramer facility (Emory University, Atlanta, GA, USA).