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. 2017 Nov 29;7:16585. doi: 10.1038/s41598-017-16828-x

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© The Author(s) 2017

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

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Gating strategy to determine pSTAT1 expression on different PBMC subsets. Based on the combination of side (SSC) and forward (FSC) light scatter properties of the acquired events, lymphocyte and monocyte gates were selected (a). T lymphocytes were identified by gating on CD3+ cells (b), then, they were transferred to a new dot plot and were analyzed by a specific antibody against CD8. T CD8+ lymphocytes were identified as CD3+ and CD8+ cells and T CD4+ lymphocytes as CD3+ and CD8- cells (c). In a new dot, monocytes were identified as CD14+ and CD3- cells (d). Overlay histograms from a representative patient that depict CD4+, CD8+ T lymphocytes and monocytes expressing pSTAT1 in the different cells stimulation conditions: unstimulated cells (black line), IFNβ stimulated cells (red line), IFNβ stimulated cells in presence of B18R inhibitor (green line), IFNβ stimulated cells in presence of NAbs positive serum (blue line), IFNβ stimulated cells in presence of NAbs negative serum (blue dashed line) and isotype control (shaded histograms) (e). PBMC: peripheral blood mononuclear cells, FSC: forward scatter, SSC: side scatter, PE-Cy7: phycoerythrin-cyanine, PerCP: peridin chlorophyll protein, Alexa488: Alexa Fluor-488, APC: allophycocyanin.