Flow Cytometric Analysis of Cytokine Expression

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)]}.