Cd4 v 500 rpa t4

Harvested cells were first washed with PBS, then labeled with 1000 times diluted Zombie NIR™ dye (Biolegend, San Diego, CA, USA) at room temperature for 20 min and washed with 0.1%BSA/PBS. For surface staining, cells were incubated with antibody mix at 4 °C for 15–20 min, then washed and resuspended with 1%PFA/PBS. For intracellular cytokine stainings, after surface staining, CytofixCytoperm™ (BD) was used to permeabilize cell membranes. Staining results were acquired either on CyAn ADP cytometer (Dako Cytomation) or LSRFortessa (BD); analysis was done using FlowJo software and R.
The following antibodies were used in this study: CD3-PB (clone UCHT1, BD), CD3-BV510 (UCHT1, BD), TCR γδ-APC (11F2, Miltenyi Biotec, Bergisch Gladbach, Germany), TCR Vγ9-PC5 (IMMU 360, Beckman Coulter, Brea, CA, USA), TCR Vδ2-FITC (IMMU 389, Beckman Coulter), CD4-V500 (RPA-T4, BD), CD4-BV510 (RPA-T4, BD), CD8-PC7 (SFCI21Thy2D3, Beckman Coulter), CD56-PE-CF594 (NCAM16.2, BD), CD69-PE (FN50, BD), IFNγ-V450 (B27, BD), TNFα-FITC (MAb11, BD), IL-17a-PE (eBio64DEC17, eBioscience). Dead cells were excluded (negative for Zombie NIR) and gated on CD3+ lymphocytes (Supplementary Figure S1). Gating CD3+Vγ9+ lymphocytes identify the vast majority of Vγ9Vδ2 T cells in adult blood [21 (link)].