Live dead fixable aqua dye

Peyer’s patches were collected from the small intestine after flushing the fecal contents. Peyer’s patches and spleens were gently smashed into single-cell suspensions. Both Peyer’s patches and splenocytes were stained with Live/Dead Fixable Aqua dye to exclude dead cells, and then stained for anti-CD3 (clone 17A2, Biolegend, San Diego, CA), anti-CD4 (clone RM4-5, Biolegend), anti-CD8 (clone 53-6.7, Biolegend), anti-CD19 (clone 6D5, Biolegend), anti-NK1.1 (clone PK136, Invitrogen), anti-CD11c (clone N418 , Biolegend), anti-CD11b (clone M1/70, BD Biosciences, Franklin Lakes, NJ), anti-Gr-1 (clone RB6-8C5, Biolegend) anti-F4/80 (clone BM8, Biolegend). Accucheck Counting Beads (Thermo Fisher Scientific, Waltham, MA) were added after staining to calculate the absolute number of cells per spleen. Samples were run on an LSRII flow cytometer (BD Biosciences) and data were analyzed using FlowJo software (Version 9.9.6 TreeStar, Ashland, OR).

DLN cells (10⁶/ml) were stimulated in the presence and absence of PMA (50 ng/ml) plus ionomycin (500 ng/ml) for 1 h before being incubated with 10 μg/ml Brefeldin A (Sigma-Aldrich, UK) for 5 h at 37°C with 5% CO₂. Live cells were discriminated by the LIVE/DEAD fixable aqua dye (BioLegend, UK) and phenotypic markers labelled using FITC-conjugated anti-CD49b (BioLegend, UK), APC-conjugated anti-γδ TCR (eBioscience, UK) and biotin-anti TLR4 antibodies before the cells were fixed and permeabilized according to BioLegend protocols. Pacific Blue-conjugated streptavidin was used for detection of biotinylated antibodies. Cytokines were stained using PerCP-Cy5.5–conjugated anti–IL-17A (BioLegend, UK) or PE-conjugated anti–IL-22 antibodies (R&D Systems, UK) for 30 minutes prior to analysis by flow cytometry, with gating according to appropriate isotype controls ^{[19 (link), 23 (link)]}.