αil 17a

For analysis of mouse cell phenotypes, the following monoclonal antibodies were used: α-CD3 (clone: 145-2C11), α-CD4 (RM4-5), α-CD8α (53–6.7), α-CD25 (PC61), α-CD44 (IM7), α-CD62L (MEL14), α-CD90.1 (OX-7), α-CD40L (MR-1), α-CD11c (HL3), α-CD11b (M1/70), α-Gr1 (RB6-8C5), α-Ly6C (HK1.4), α-Ly6G (1A8), α-F4/80 (BM8), α-IFN-γ (XMG1.2), α-IL-17A (TC11-18H10), α-GM-CSF (MP1-22E9; all from BioLegend) and α-IL-22 (AM22-3; provided by J.C. Renauld). For staining of lipid rafts, biotin-conjugated cholera toxin subunit B (Molecular Probes) was used together with fluorochrome-conjugated streptavidin (BioLegend). Dead cells were always excluded by staining with 7-AAD (BioLegend) or 4,6-diamidino-2-phenylindole (Sigma). All antibodies were used at 1 μg per 10⁶ cells. For intracellular cytokine staining, cells were stimulated for 5 h with phorbol 12-myristate 13-acetate (10⁻⁷ M; Sigma) and ionomycin (1 μg ml⁻¹; Sigma), in the presence of brefeldin A (10 μg ml⁻¹; Sigma) for the last 3 h of culture. Cells were fixed with 4% (wt/vol) paraformaldehyde and permeabilized with 0.5% (wt/vol) saponin (Sigma). Eight-colour staining was performed with the appropriate combinations of antibodies conjugated to fluorochromes. Samples were acquired on a FACSCanto II (BD Biosciences) and analysed with FlowJo software (TreeStar) and when necessary using the Boolean gating strategy65 (link).