Anti ifnγ pacific blue

Draining lymph node (DLN) cells (10⁶/ml) were incubated ± 50 ng/ml PMA plus 500 ng/ml ionomycin for 1 h before addition of 10 μg/ml Brefeldin A (Sigma–Aldrich, UK) for a further 5 h at 37 °C with 5% CO₂. Live cells were discriminated by the LIVE/DEAD fixable aqua dye (Invitrogen) and phenotypic markers were labelled using anti-CD4-PerCP, anti-CD8-FITC or anti-γδ-PE (BioLegend) antibodies before the cells were fixed and permeabilised using BioLegend protocols. Cells were then labelled using anti-IFNγ-Pacific Blue or anti-IL-17A-APC (BioLegend) antibodies for 30 min prior to flow cytometry and gated according to appropriate isotype controls as described previously [7] (link). IL-12p40 and IL-17 levels in serum or DLN, bmM and peritoneal exudate cell (PEC) supernatants were detected by ELISA using kits from BioLegend as described previously [7] (link) whilst levels of IL-1β were determined by ELISA using kits from eBioscience according to the manufacturer's recommendations.

During necropsy spleens were collected in Iscove’s Dulbecco’s Medium (IMDM, Lonza) supplemented with 5% FBS and P/S/G for direct preparation of single cell suspensions using 100 µm strainers (Falcon). Erythrocytes were removed from single cell suspensions by treatment with red blood cell lysis buffer (Roche diagnostics). For intracellular cytokine staining, splenocytes were stimulated with 5 µM synthetic peptide (epitope NP_366–374: ASNENVEIM [Fig. S2] or ASNEMMETM [Fig. 4]) or 1 µg/250,000 cells recombinant HA protein from H5N1 influenza virus A/Vietnam/1203/04 or A/Indonesia/5/05 (Protein Sciences) in IMDM supplemented with GolgiStop and incubated for 6 h at 37 °C. Mock-treated splenocytes and splenocytes stimulated with 50 ng/ml PMA (Sigma-Aldrich) and 0.5 µg/ml ionomycin (Sigma-Aldrich) served as appropriate negative and positive controls. After stimulation, splenocytes were incubated with fluorochrome-labeled antibodies to CD3e^APC-Cy7 (BD Pharmingen), CD8b^FITC (BD Pharmingen), CD4^PerCP (BD Pharmingen) and viable cells were identified with Aqua LIVE/DEAD (Invitrogen). Subsequently, cells were fixed and permeabilized using BD Cytofix/Cytoperm^TM Plus (BD Biosciences), and incubated with anti-IFN-γ^PacificBlue (Biolegend). Samples were acquired on a FACS Canto II and data was analyzed as described previously^{55 (link),59} using FACS Diva software (BD Biosciences).