Anti tcr vα7.2 pe 3c10

The percentage of cells producing IFN-γ was measured by flowcytometry using various stimuli. For each condition, 500,000 cells were stimulated in a 24 wells plate with IL-12 (0.25 ng/ml, Miltenyi), IL-18 (50 ng/ml, MBL) and IFN-α (2500 IU/ml, Intron-A, Merck). For all conditions, cells were stimulated for 16 hours at 37°C at 5% CO₂. Brefeldin A (10 μg/ml, Sigma) was added and the cells were incubated for another 3 hours. Cells were stained with anti-CD3-PerCp-Cy5.5 (UCHT1), anti-CD4-APC-H7 (SK3, both BD biosciences), anti-CD69-PE-Cy7 (TPI.55.3), anti-CD161-PB (HP-3G10, both eBiosciences), anti-TCR Vα7.2-PE (3C10, Biolegend), anti-CD56-APC (N901, Beckman) and Live/ dead marker (Aqua, Life technologies). Cells were fixed, permeabilized and stained with anti-IFN-γ-FITC (25723.11, BD Biosciences). Cytokine-producing cells were detected by flow cytometry (Canto-II, BD). Quadrants were set on low or absent expression on lineage negative cells. Data was analyzed using FlowJo version 10.1 (Tree Star Inc).

Peripheral blood mononuclear cells (PBMC) were isolated from venous blood by (Ficoll-Paque^™ plus, Amersham) and frozen at -150°C. PBMC were thawed and washed with RPMI 1640 (Lonza) with 10% FCS (Lonza). For flow cytometry, 500,000 PBMC were used for each staining. Cells were stained with anti-CD38-PerCp-eFluor710 (HB7), anti-CD3-PE-Cy7 (UCHT1), anti-CD161-Pacific Blue (HP-3G10, all eBiosciences), anti-CD4-APC-H7 (SK3, BD Biosciences), anti-TCR Vα7.2-PE (3C10, Biolegend), anti-CD56-APC (N901, Beckman) and live/dead marker (Aqua, Life technologies) for 20 minutes at 4°C in the dark. Positivity for CD38 was determined by setting the gates using internal negative cells. Cells were washed and marker expression was detected by flow cytometry (Canto-II, BD). MAIT cells were defined as CD3⁺CD161⁺Vα7.2⁺ cells within the lymphocyte gate and NK cells were defined as CD3^-CD56⁺ cells within the lymphocyte gate. Data was analyzed using FlowJo version 10.1 (Tree Star Inc).