Perm wash reagent

Cells were stained in 'FACS buffer' (PBS with 0.1% sodium azide, 2% FBS and 1 μM EDTA) with antibodies to TCRγδ (GL3), TCRβ (H57-597), IL17A (eBio17B7), Ccr6 (140706), IL7Rα (A7R34), CD3ε (clone 145-2C11), CD11b (clone Mac-1), Vγ4 (clone UC3-10A6), CD90.2 (30-H12), S1pr1 (R&D, MAB7089, clone 713412), anti-CD169 (clone MOMA-1 and clone Ser4); anti-scart2 antibody was kindly provided by Dr. Klaus Karjalainen. Molecular Probes Monoclonal Antibody Labeling Kits (Invitrogen) were used to directly conjugate antibody to Alexafluor647 or Pacific Blue dyes. During analysis, singlets were gated based on peak FSC-H/FSC-W and SSC-H/SSC-W. These gates encompassed more than 90% of total events and were set sufficiently wide to include singlet events of variable size while avoiding the main doublet peak.
To detect IL-17A, cells were stimulated for 3 hr with 50 ng/ml phorbol 12-myristate 13-acetate (PMA, Sigma) and 1 µg/ml Ionomycin (I, EMD Biosciences) or 3 hr with 10 ng/ml IL1β and 10 ng/ml IL23 in Golgi plug (BD Biosciences) at 37°C, stained for surface antigens, treated with BD Cytofix Buffer and Perm/Wash reagent (BD Biosciences), and stained with anti-IL-17A.

Each mouse lymph nodes and spleen were minced separately and passed through a 70 μm cell strainer to obtain single-cell suspensions. Single cells from the draining lymph nodes were used for the detection of innate immune cells and were stained with the following monoclonal antibodies: F4/80-PerCP-Cy 5.5, CD11c-PE, CD11b-APC, and Ly6G-FITC (BD Biosciences, USA). The γδT and Th17 cells from the spleen samples were detected with the following fluorescent antibodies: CD45-PerCP-Cy 5.5, γδT-PE, CD4-APC, and IL-17A-PE (BD Biosciences, USA). For the surface antigen staining, cells were incubated with the antibodies for 30 min at room temperature (RT) after washing the cells with PBS. To stain the intracellular cytokines, the cells were stimulated with a cell stimulation cocktail (eBiosicence) for 4 h. After that, the cells were stained for surface antigens and then fixed with BD Cytofix buffer, permeabilized by using Perm/Wash reagent (BD Biosciences) and then stained with anti-IL-17A antibodies. The cells were acquired using an LSR II Flow Cytometer (BD Biosciences), and the data were analyzed using the Flow Jo software version 7.0 (Tree Star, California, USA).