10.6084/m9.figshare.c.3737758_D4.v1 Sean Wattegedera Sean Wattegedera Yolanda Corripio-Miyar Yolanda Corripio-Miyar Yvonne Pang Yvonne Pang David Frew David Frew Tom McNeilly Tom McNeilly Javier Palarea-Albaladejo Javier Palarea-Albaladejo Colin McInnes Colin McInnes Jayne Hope Jayne Hope Elizabeth Glass Elizabeth Glass Gary Entrican Gary Entrican MOESM4 of Enhancing the toolbox to study IL-17A in cattle and sheep Springer Nature 2017 intracellular IL -17A FITC cells Forward Scatter Height intracellular cytokines K-L vs Vioblue channel vs FSC-Area SSC-H FSC-H Table  3 ovine T cell subsets  section MOESM 4 intracellular cytokine staining APC WC cell surface markers IFN -γ two-colour cell phenotyping study IL -17A 4. Gating strategy PBMC cell marker FMO Quadrant region boundaries antibody controls representative animal cytokine IL -17A I-J IFN -γ. Activated T cell subsets Side Scatter Height 2017-04-08 05:00:00 Journal contribution https://springernature.figshare.com/articles/journal_contribution/MOESM4_of_Enhancing_the_toolbox_to_study_IL-17A_in_cattle_and_sheep/4832071 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.