Anti cd161 pe

We stained cells with appropriate antibodies, which were analyzed using a BD FACSCalibur flow cytometer (BD Biosciences, Franklin Lakes, NJ). Data were analyzed using CellQuest software ver 6.0 (BD Biosciences). We stained GBM cell lines using the following mouse anti-human antibodies: anti-CD112-PE (R2.525) and anti-CD50-PE (TU41)—BD Biosciences; anti-MIC-A-PE (AMO1) and anti-MIC-B-PE (BMO1)—MBL; anti-ULBP-1-PE (170818), anti-ULBP-2-PE (165903), and anti-ULBP-3-PE (166510)—R&D Systems (Minneapolis, MN); and anti-HLA-ABC-PE (W6/32), anti-HLA-E-PE (3D12), anti-CD48-PE (156-4H9), anti-CD155-PE (2H7CD155), anti-CD54-PE (HA58), and anti-CD102-PE (CBRIC2/2)—Thermo Fisher Scientific (Waltham, MA).
The expanded NK cells were stained with the following antibodies: anti-CD3-FITC (UCHT1), anti-CD56-PE-Cy5 (B159), anti-CD11a-PE (HI111), anti-CD16-PE (3G8), anti-DNAM-1-PE (DX11), anti-NKp30-PE (P30-15), anti-NKp44-PE (P44-8.1), anti-NKp46-PE (9E2/NKp46), and anti-CD161-PE (NKR-P1A)—BD Biosciences; anti-CD158a-PE (EB6Bf) and anti-CD159a-PE (NKG2A)—Beckman Coulter (Pasadena, CA); anti-NKG2D-PE (149810)—R&D Systems; anti-CD2-PE (RPA-2.10), anti-CD4-PE (OKT4), anti-CD8-PE (SK1), anti-CD244-PE (C1.7), anti-CD94-FITC (DX22), and anti-CD158b-PE (GL183)—Thermo Fisher Scientific.

The following anti-human monoclonal antibodies were used for surface phenotype and intracellular cytokine staining: anti-CD4-fluorescein isothiocyanate (FITC); anti-CD4-phycoerythrin (PE); anti-CD161-PE; anti-CCR6-PE; anti-CD45RO-PE; anti-CD147-peridinin chlorophylla protein cyanine 5.5 dies (Percp–cy5.5); anti-interferon (IFN)-γ-FITC (all from BD Biosciences); and anti-IL-17A-allophycocyanin (APC; eBiosciences). Anti-mouse monoclonal antibodies included: anti-CD4-Percp; anti-IL-17A-APC; and anti-IFN-γ-FITC (all from BD Biosciences). Appropriately conjugated IgG antibodies were used as isotype controls. Cells were acquired on a FACSCalibur flow cytometer (BD Biosciences) and analyzed using Cell Quest software (BD Bioscience) and FlowJo 7.6.1 software (Tree Star).

Fluorochrome-conjugated monoclonal antibodies specific for human antigens were used anti-CD4-FITC (BD Bioscience, catalog 561005), anti-TCR Vα7.2-APC (BioLegend, catalog 351708), anti-IFN-γ-APC (BD Bioscience, catalog 562017), Anti-CD3-FITC (Bioscience, catalog 555339), Anti-CD161-PE (BD Bioscience, catalog 556081). Intracellular staining was performed using eBioscience™ Intracellular Fixation & Permeabilization Buffer Set (ThermoFisher) according to the manufacturer’s protocols.

MAIT cells were identified as CD161^hiTCR Vα7.2⁺ cells among CD3⁺ T cells. To detect surface markers, the following antibodies were used: anti-CD3-APC-Cy7 (BD Biosciences, San Diego, California, USA. Clone: OKT3), anti-CD161-PE (BD Biosciences. Clone: HP-3G10), and anti-TCR Vα7.2-BV421 (BD Biosciences. Clone: 3C10). For fluorochrome-labeled inhibitors of caspases (FLICA) caspase-1 detection, FLICA staining was conducted in accordance with the manufacturer’s instructions (Bio-Rad, Hercules, CA, USA). Cells were incubated with the FLICA caspase-1 reagent for 1 hour at 37 C and then cells were washed for downstream surface markers staining and intracellular anti-capase-3-Alexa 647 (BD Biosciences. Clone: C92-605). Data were acquired on a BD FACSCanto II flow cytometer (BD Biosciences), and further analyzed using FlowJo (Ashland, OR, USA) Tree Star software.