Anti cd161 fitc

EDTA-treated whole blood was incubated using the corresponding monoclonal antibodies: anti-CD3-PC5.5 and anti-CD4-APC-A750 (all from Beckman Coulter, Miami, FL, USA); anti-TCRγδ-PE, anti-NKG2D-PCy7 and anti-CD8-APC (all from BDBiosciences, Franklin Lakes, NJ, USA); and anti-CD56-FITC and anti-CD161-FITC (all from BioLegend, San Diego, CA, USA). Innate subsets were analyzed by flow cytometry using a Dx-Flex Cytometer and Kaluza Software (Beckman Coulter, Miami, FL, USA).
NK and NKT cells were considered CD3-CD56+ and CD3 + CD56+, respectively, gated from lymphocytes. MAIT cells were considered Vα7.2+ and CD161+ gated from CD3 + αβTCR+ T cells. MAIT cells expressing CD4 and CD8 were gated from CD3 + CD4+ or CD8+ lymphocytes, respectively, expressing both Vα7.2 and CD161.

For flow cytometry analysis, tumor and blood samples were stained with a cocktail of monoclonal mouse anti-human conjugated antibodies (mAbs): anti-CD45-PercP (clone HI30), anti-CD3-PercP (HIT3a), anti-CD3-APC (UCHT1), anti-CD19-PE (HIB19), anti-CD15-PE (HI98), anti-CD161-FITC (HP-3G10), anti-CD4-FITC (OKT4), anti-CD8-PE (HIT8a), anti-HLA-DR-APC (L243), anti-CD127-PE-Cy7 (A019D5), anti-CD1c-APC-Cy7 (L161), anti-CD163-PE (GHI/61), anti-CD206-APC-Cy7 (15–2), anti-PD-1-FITC (EH12.2H7), anti-PD-L1-APC (29E2A3), anti-CTLA4-PE (L3D10), anti-CD69-APC-Cy7 (FN50), anti-Tim3-APC (F38-2E2), anti-IL-8-APC (E8N1), anti-IFN-γ-PE (4S.B3), anti-IFN-γ-APC-Cy7 (4S.B3), anti-Granzyme B-FITC (QA16A02), anti-IL-1β-FITC (JK1B-1), anti-IL-2-PE-Cy7 (MQ1-17H12), anti-IL-6-APC (MQ2-13A5), anti-IL-17-FITC (BL168), anti-IL-23/IL-12-PE (C11.5), anti-TGF-β-APC (TW4-6H10), all from Biolegend; anti-IDO-PE (eyedio) and anti-IL-10-FITC (BT-10), both from eBioscience; anti-CD25-PE (MEM-181) and anti-CD11b-FITC (LT11) from ImmunoTools.
The antibodies used for immunofluorescence were: mouse monoclonal anti-human CD8 (32-M4) from Santa Cruz Biotechnology and rabbit polyclonal anti-human HLA-DRA from Sigma Aldrich.