Anti il 9

Supernatants from cell cultures were collected after activation under various conditions and secreted cytokines in the supernatants were measured by ELISA kits with purified coating and biotinylated detection antibodies: anti-IL-17, anti-IFN-γ (R & D systems), anti-IL-9, anti-IL-22, anti-IL-1β (e Bioscience) and anti-IL-4 (BD Bioscience).

For intracellular cytokine staining cultured cells were restimulated with PMA (50 ng/mL, Sigma Aldrich) and Ionomycin (1 μg/mL, Invitrogen, Life technologies) in the presence of Monensin (2.5 mM, Sigma Aldrich) for 5 h. To measure the cell viability (>80%) the cells were stained with Annexin-V FITC and 7-AAD according to the manufacturer's protocol (BD Bioscience). The cells were fixed with 4% paraformaldehyde and permeabilized with 0.1% saponin-containing buffer before washing and staining in different combinations with anti-CD4 (Beckman Coulter, Krefeld, Germany), anti-IL-17A, anti-IL-22, anti-IL-9, anti-IL-8, anti-AHR, anti-RORγt (ebioscience), and anti-IFN-γ (BD Bioscience). All dotplots and histograms shown refer to gated CD4⁺ T cells. Data were acquired on FACSCantoII (BD Bioscience) and were analyzed with FlowJo Software (Tree Star Inc., Ashland, OR, USA).

We used the following antibodies and reagents: anti-CD4 (clone: GK1.5)-FITC, -PerCP-Cy5, or -APC, anti-CD8 (clone: 53-6.7)-FITC, -PerCP-Cy5, or -APC, anti-IFN-γ (clone: XMG1.2)-APC, anti-CD44 (clone: IM7)-APC, anti-BrdU (clone: Bu20a)-PE, 7-AAD, anti-IL12 (clone: C15.6)-PE, anti-IL4 (clone: 11B11)-PE, anti-IL22 (clone: Poly5164)-PE, anti-IL10 (clone: JES5-16E3)-PE, anti-IL9 (clone: MH9A4)-PE, and anti-CD69 (clone: H1.2F3)-PE (all from eBioscience); anti-caspase-3 (clone: C92605)-FITC (From BD Biosciences); anti-FasL (SC6237) (Santa Cruz Biotechnology); and IgG-Fab2 (clone: 4412) (Cell Signaling).