Gata3 l50 823

Cells from small intestinal lamina propria (siLP) and large intestinal lamina propria (LiLP, including caecum and colon) were prepared as previously described [28 (link)]. Single-cell suspensions were stained with CD16/32 (2.4G2) and with fluorochrome-conjugated antibodies against any combination of surface antigens. Prior to fixation, Live/Dead Fixable Blue Cell Stain Kit (Invitrogen) was used to exclude dead cells. For intracellular cytokine staining, isolated cells from lamina propria were stimulated for 2 h with phorbol 12-myristate 13-acetate (PMA) (50 ng/ml) and ionomycin (2.5 μg/ml) or IL-23 (5 ng/ml) in the presence of Brefeldin A (1 μg/ml) (Sigma). For examination of transcription factors and intracellular cytokines, cells were subsequently treated with the FOXP3 staining kit (eBioscience) in accordance with the manufacturer's instructions and stained for 20 min at room temperature with fluorochrome-conjugated antibodies. Antibodies specific to mouse CCR6 (140706) and GATA3 (L50-823) were purchased from BD Biosciences; TCR-β (H57), TCR-γδ (GL3), CD11b (M1/70), CD19 (6D5), Gr-1(RB6-8C5), Ly76 (TER-119), CD127 (A7R34), CD45.1 (A20), CD45.2 (104), Sca-1 (E13-161.7), c-Kit (2B8), NKp46 (29A1.4) were purchased from BioLegend; RORγt (B2D), T-bet (4B10), IL-22 (IL22JOP) and IFNγ (XMG1.2) were purchased from eBioscience. Results were analyzed with FlowJo software (Tree Star).