Fitc anti vδ2 tcr

Monophosphoryl lipid A (MPLA) and the imidazoquinoline compound CL097 were purchased from InvivoGen, IL1β from Biosource, IFNα2b from ProSpec, IFNγ from Immunotools, tumor necrosis factor (TNF)α from Peprotech, polyinosinic:polycytidylic acid (polyIC) and prostaglandin (PG)E₂ from Sigma-Aldrich, whereas IL6 was a kind gift from S. Rose-John (Institute of Biochemistry, University of Kiel, Kiel, Germany).
Following monoclonal antibodies (mAb) were used: PE anti-HLA-DR, Alexa Fluor®700 anti-CD3, eFluor^TM450 anti-CD16, PE-Cy7 anti-CD14, allophycocyanin-efluor780 anti-CD4 (eBioscience/Affimetrix GmbH), PE-Cy7 anti-CD56 (Beckman Coulter), FITC anti-Vδ2 TCR, allophycocynin anti-CD107a, BV570 anti-CD8α (Biolegend Inc.), allophycocyanin anti-CD14, allophycocyanin anti-CD25, PE anti-CD127, PE-Cy7 anti-CD33, FITC anti-CD11b (Becton–Dickinson) and PE anti-CCR7 (R&D System).

Multicolor flow cytometry gating strategy was applied to analyze various cell populations simultaneously. Expanded cells were co-stained with 6 different antibodies and analyzed with LSRII flow cytometer (BD Bioscience, San Jose, CA). Briefly, cells were re-suspended in 100 μl MACS buffer (Miltenyi Biotec, Auburn, CA) and stained with antibody cocktail in the dark at 4°C for 15 minutes. Washing twice with MACS buffer was performed before and after cell staining. The antibody cocktail contains: FITC-conjugated anti-Vδ2 TCR (Biolegend, San Jose, CA), APC-conjugated anti-CD3 (Biolegend), PE conjugated anti-CD4 (BD), PE-CyC7-conjugated anti-CD56, APC-CyC7-conjugated anti-CD16, V450-conjugated anti-CD8a Beckman Coulter (Fullerton, CA). The antibody cocktail used for Treg analysis contains CD4-PE, CD25-APC (BD Bioscience, San Jose, CA), and CD127-FITC (eBioscience) to detect CD4+CD25+CD127- Treg population. The gating strategies used in the current study are included in S1 and S2 Figs. Cell populations were also analyzed with one- or two-color flow cytometry in Accuri C6 flow cytometer (BD). FITC-anti-Vδ2 TCR, APC-anti-CD3, and PE-anti-CD3, and APC-anti-CD56 (Biolegend) were used.