MOESM4 of Cytokine production and phenotype of Histomonas meleagridis-specific T cells in the chicken

Additional file 4. Gating strategy for lymphocytes from spleen and liver in multicolor flow cytometry. For lymphocytes subjected to intracellular IFN-γ staining (A) and PrimeFlowTM RNA Assay (Thermo Fisher Scientific) staining for IL-13 mRNA (B) a time gate as well as FSC-H/FSC-W and SSC-H/SSC-W doublet discrimination gates were applied consecutively. Lymphocytes were then selected within a FSC-A/SSC-A plot followed by a dead cell exclusion gate using the Fixable Viability Dye eFluor® 780. (A) Frequencies of IFN-γ+ cells within CD4+, CD8β+ and CD4−CD8β− subgates were determined. (B) Percentages of IL-13 mRNA+ cells were determined within total live lymphocytes after excluding cells stained with putative dye aggregates in the CD4/CD8β plot. The gating strategy is shown for splenocytes from representative experiments and was applied for both organs from all birds.