MOESM2 of Enhancing the toolbox to study IL-17A in cattle and sheep

Additional file 2. Gating strategy used for the evaluation of commercial antibodies to bind intracellular recombinant bovine and ovine IL-17A in fixed cells. Cells were acquired for flow cytometric analyses using the MacsQuant flow cytometer and analysed using the MacsQuantify Software. 20 000–50 000 events were collected and the following gating strategy was followed. Cells in the plot of Forward Scatter-Area (FSC-A) against the high dynamic range over time (HDR-T) are gated in P1 to exclude any non-specific artefacts (A). The P1/P2 gate represents Side Scatter-Area (SSC-A) plotted against FSC-A set to identify the main cell population and exclude debris (B). Single cells were gated (P1/P2/P3) using FSC-Height (H) vs FSC-A for doublet discrimination (C). Finally, the cells of interest were identified in the phycoerythrin or alexafluor 488 channel vs SSC-A (P1/P2/P3/P4) where regions were set using the isotype or equivalent control for each CHO cell line to establish threshold gates (D). Overlaying histogram plots of phycoerythrin or alexafluor 488 using (P1/P2/P3) gating strategy selecting for all cells in the region (equivalent to cells above and below region boundary in plot D) (E) were used to compare anti-IL-17A antibodies with appropriate isotype or equivalent controls presented in Figure 4. Gated percentage numbers above the region boundary (P1/P2/P3/P4) and median fluorescence region values (P1/P2/P3) were measured for each antibody in the relevant fluorochrome channel phycoerythrin or alexafluor 488. Delta median fluorescence intensity (deltaMFI) was calculated by deducting the median fluorescence region value for mab isotype control or pab control from the anti-IL-17A antibody value for the appropriate fluorochrome channel. The summarised data are presented in Additional file 3.