Cd161 bv421

Anti-Bcl-2 PE, CD3-FITC, CD8-APC, CD127-BV510, IFNγ-APC, RORγt-BV421, TNF-PE were from BD Biosciences. Anti-CD4-PerCP, CD8-BV510, CD127-PECy7, CD161-BV421, -FITC, and -PE, IFNγ-APC, Ki-67-AF488 and -BV510, TNF-BV421, and Vα7.2 PECy7 were from Biolegend. Anti-PLZF APC was from R&D Systems. CellTrace Violet (CTV) cell proliferation kit, Live/Dead Aqua and Near Infrared fixable cell stain were from Life Technologies. MR1-5-OP-RU tetramer -APC, -BV421, and -PE were from NIH Tetramer Core Facility, Emory University. Staining with the MR1 5-OP-RU tetramers was performed for 40 min at room temperature (RT) (6 (link)) before proceeding to the surface and intracellular staining with other mAbs. Cell surface and intracellular staining for TFs, cytokines, and cytotoxic molecules were performed as previously described (10 (link)). Samples were acquired on an FACS Canto II (BD Biosciences) equipped with 405, 488, and 633 nm lasers or CytoFLEX (Beckman Coulter) equipped with 405, 488, and 638 nm lasers. Data including the compensation platform were analysed using FlowJo v.10 (BD Biosciences).

Cells were stained with the following antibodies: anti-human ROR-γt-BV421 (BD Biosciences) (1:20), anti-human CCR6-Alexa 647 (BD) (1:20), anti-human IL-1R1-PE (RnD) (1:20), anti-human CD4-PECy7 (Beckman Coulter) (1:100), CD161-BV421 (Biolegend, San Diego, CA, USA) (1:40), anti-human CD3 FITC (Miltenyi) (1:100). Samples were analysed using Cytoflex cytometer (Beckman Coulter, Brea, CA, USA) and analysed using FlowJo-10 software version 10.3.0.