Anti rorγt pe

Cells were labelled with flow antibodies for 15 minutes in the dark in PBS containing 2% BSA. Labelled cells were washed twice and resuspended in PBS containing 2% BSA. The prepared samples were analyzed using an LSR-II flow cytometer (BD Bioscience) or sorted using an Aria II cell sorter (BD Bioscience). Anti-CD19-PeCy7 (561739), anti-CD3-PeCy7 (552774), anti-NK1.1-BV421 (562921), anti-NKp46-FITC (560756), anti-NKp46-AF647 (560755), anti-CD49b-PE (553858), anti-CD49a-PerCP-Cy5.5 (564862), anti-CD62L-APC (553152), anti-T-bet-APC (561264), anti-CD45.2-AF700 (560693), anti-CD45.2-FITC (553772), anti-Gata3-AF647 (560068), anti-RORγt-PE (562607), anti-CD127-V450 (561205), anti-CD117-PE (553869), anti-LPAM-1-APC (562376), anti-Flt3-BV421 (566292), anti-Ly-6A/E-APC (565355), and anti-CD122-PE (553362) were purchased from BD Bioscience. Anti-IFN-γ-AF-700 (505823) and anti-CD25-Pacific Blue (102022) were purchased from Biolegend. Anti-CD253-APC (17-5951-82), anti-Eomes-PE (12-4875-82), and anti-CD127-PerCP-Cy5.5 (45-1271-80) were purchased from eBioscience.

Cell surface and intracellular phenotypes were determined by flow cytometry using the following reagents: anti‐CD4‐Alexa 488 (0.2 μg/mL, RM4‐5); anti‐ROR‐γT‐PE (2 μg/mL, Q31‐378); anti‐IL‐4‐PE ((2 μg/mL, BVD4‐1D11) BD Biosciences, UK); anti‐CD25‐eFluro450 (0.2 μg/mL, eBio3C7); anti‐IL‐17‐APC (0.2 μg/mL, eBio17B7), anti‐CD69‐PE (0.2 μg/mL, H1.2F3), and anti‐IFN‐γ‐PEcy7 ((0.2 μg/mL, XMG1.2) eBioscience, UK). For intracellular cytokine staining, cells were blocked with 200 μg/mL of rat serum (Sigma, UK) and fixed and permeabilized using Foxp3 staining kit (eBioscience) according to manufacturer's instructions. Data were acquired using FACS Canto‐II (BD) and analyzed by Flowjo software 8.7.1 (Treestar, USA). The following gating strategy was used (see also Supporting Information Fig. 1: (i) a ‘life’ gate was set on the FSC/SSC plot; (ii) a gate was set on CD4+ cells, based on an isotype‐matched control plot; (iii) marker of interest‐positive cells were gated on the basis of a fluorescence‐minus‐one plot – unstained and isotype‐matched control samples were used to exclude nonspecific binding of mAbs used.