, 1995). After incubation, cells were washed twice with FACS buffer and were either used for intracellular staining or fixed with a solution of 2% paraformaldehyde in PBS. Incubation with primary antibodies to MHC I (Salomonsen et al., 1987) and MHC II (Kaufman et al., 1990) was followed by Alexa-647 conjugated goat anti-mouse antibody (Life Technologies). Secondary antibody alone or unconjugated goat anti-mouse antibody (Life Technologies) was used as an unstained control for surface MHC staining. Intracellular staining JQ1 chemical structure was carried out as described previously (Ariaans et al., 2008). Briefly, splenocytes from challenged birds or non-infected controls were seeded in a 96-well round-bottom plates (Nunc)
at 106 cells/well in a final volume of 200 μl of culture media or culture media supplemented with the different stimuli at the concentration described in the ELISpot technique (except PMA which was used at 50 ng/ml). Cells
were cultured using the conditions described above for ELISpot assays (24 h culture). Selleckchem Epacadostat For intracellular staining, during the last 2 h of culture, cells were treated with Brefeldin A according to the manufacturer’s instructions (Cytofix/Cytoperm™ Plus Fixation/Permeabilization kit, BD Biosciences). To avoid non-specific binding signal, we preincubated cells with Cytofix/Cytoperm™ buffer containing 2% normal mouse serum and further staining steps involved Cytofix/Cytoperm™ washing buffer containing 1% normal mouse serum (Biosource). To confirm the specificity of the anti-IFNγ antibody EH9 we also employed a validated anti-IFNγ [mAb80 (Ariaans et al., 2008)]. Purified fractions of both antibodies were conjugated using Alexa Fluor® 647 monoclonal antibody labeling kit (Molecular Probes) according to the manufacturer’s selleck screening library instructions. A mouse isotype matched control antibody IgG1 Alexa Fluor® 647 (Life Technologies) was employed at the same concentration as EH9 and Mab80. For analysis, a gate on the FSC/SSC region of lymphocytes was selected and a minimum of 10,000 events were acquired on a FACSCalibur instrument using Cell Quest software (BD Becton Dickinson). Flow cytometry
analysis indicates that non-adherent CKC were not present at significant levels (data not shown). FlowJo software (TreeStar) was used to analyze flow cytometry data. A paired or unpaired t-student test or one-way ANOVA was performed using GraphPad Prism (version 6.0 for Windows, GraphPad Software, San Diego, California, USA). Screening identified two anti-chicken IFNγ antibodies (clones EH9 and AF10) which were shown by ELISA to bind recombinant chicken IFNγ and to work effectively as an antibody pair in capture ELISA (Supplementary Fig. 2A–C). We subsequently compared this antibody pair with commercially available antibodies [from Life Technologies (Ariaans et al., 2008 and Reemers et al., 2012)] in ELISpot assays.