Il 17 tc11 18h10

To label the Th1 and Th2 cytokines in serum, we used a Cytometric Bead Array Mouse Th1/Th2/Th17 Cytokine kit (BD Biosciences) following the manufacturer's instructions and using a FACS Canto II (BD Biosciences). To characterize the immune cell populations, single cell suspensions of lymphocytes were prepared from large intestine lamina propria
34, 35. Lymphocytes were stimulated for 4 h using ionomycin, phorbol myristate acetate (Sigma), and 1 μg/ml Golgistop (Sigma). Prior to flow cytometry, lymphocytes were stained with fluorescent conjugated antibodies against CD4 (RM‐4‐5, 1/800), Ifng (XMG1.2, 1/200), IL‐13 (eBIO 13a, 1/200), TCRa/b (MR5.2, 1/200) (all Fisher Scientific), CD45 (30‐F11, 1/200), CD8 (53‐6.7, 1/200), IL‐4 (11B11, 1/200) (all Biolegend, London, UK), IL‐17 (TC11‐18H10.1, 1/100) and TNF (MP6‐XT22, 1/200) (both BD Biosciences), and a live/dead marker. Data were analysed using FCAP Array v3.0 (BD Biosciences) and FlowJo (FlowJo LLC, Ashland, OR, USA) software. Statistical analysis was performed using three to five animals per genotype and three independent experiments. Significance was calculated using a three‐way full factorial fit model and a joint F‐test to assess the effects of genotype, treatment, and experiment day on the cytokine response
36.

The following FACS antibodies were purchased from BD Biosciences: CD4 (RM4-5), CD44 (IM7), Ki67 (B56), IFNγ (XMG1.2) and IL-17 (TC11-18H10). The following were purchased from eBioscience: CD73 (eBIOTY/11.8), RORγt (AKFJS9), Foxp3 (FJK-16s) and GM-CSF (MP1-22E9). For cytokine analysis, cells were cultured in complete medium (as described for T cell cultures above) with 50 ng/ml PMA and 500 ng/ml ionomycin (Sigma-Aldrich) in the presence of Golgiplug (BD Biosciences) for 3 to 4 hours followed by FACS staining and analysis. For intracellular cytokines, staining was performed using Cytofix-cytoperm kit from BD; RORγt and Foxp3 intracellular stains were performed using eBioscience Foxp3 staining kit according to manufacturer’s instructions. Prior to surface staining, cells were incubated for 20 min on ice with Ghost Dye^™ Violet 510 (TONBO biosciences, CA) to allow exclusion of dead cells from analysis performed in FlowJo.