Live dead fixable blue cell stain kit

Flow cytometry was performed using a standard protocol. Fc receptor blocking was carried out with anti-CD16/32 specific antibodies. A lineage cocktail of antibodies specific for CD3, CD45R, CD19, CD11b, TER-119, Gr-1, CD5 and FcϵRI was used for cLP ILC analyses. Live/Dead Fixable Blue Cell Stain Kit (Invitrogen) stain was used to exclude dead cells from the analysis. The cLP ILC gating strategy is outlined in our recent publications (44 (link), 45 (link)). For a complete list of the antibodies used see Table 1. A FoxP3 staining kit (ebioscience) was used for intracellular staining of cytokines and transcription factors. In case of cytokine expression analyses, cells were pre-stimulated with 100 ng/ml PMA and 2 µM ionomycin in the presence of 6 µM monensin for 3-4 hours prior to flow cytometry analysis. Samples were acquired using an LSRFortessa™ cell analyser (Becton Dickinson, USA), and all the data were analyzed using FlowJo software (Tree Star, USA).

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).