MOESM4 of Enhancing the toolbox to study IL-17A in cattle and sheep

Additional file 4. Gating strategy for the identification of activated bovine T cell subsets expressing intracellular IL-17A and IFN-γ. Activated bovine PBMC were stained for viability, cell surface markers and intracellular cytokines according to the protocol outlined in “Expression of intracellular IL-17A and IFN-γ by bovine and ovine T cell subsets section” using antibodies listed in Table 3 and acquired using an LSRFortessa™ cell analyzer (Becton–Dickinson). Cells were gated to eliminate dead cells using the Vioblue Live/Dead® Fixable Dead Cell Stain Kit, Side Scatter Height (SSC-H) vs Vioblue channel (A) and to include only single cells Forward Scatter Height (FSC-H) vs FSC-Area (FSC-A) (B). Gated single cells used for subsequent two-colour cell phenotyping and intracellular cytokine staining (C). Quadrant region boundaries were set based on isotype-matched directly conjugated antibody controls (FITC vs APC/Alexafluor 647 channels, D) and (Phycoerythrin vs APC/Alexafluor 647 channels, E) and fluorescence minus one (FMO) controls for each cell marker (WC-1, F; CD4, G; CD8β, H) and for each cytokine IL-17A, I-J and IFN-γ, K-L). Data are shown for one representative animal of four.