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. 2019 Nov 14;9:16791. doi: 10.1038/s41598-019-53069-6

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© The Author(s) 2019

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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CysA2 is an immunogenic protein capable of activate dendritic cells and macrophages. Murine dendritic cells and macrophages were incubated with CysA2 for further CD86 expression analysis. Polymyxin was used to neutralize any LPS contamination on CysA2 protein. LPS was used as a positive control of immune cells activation. (a) The gate strategy for the analysis of dendritic cells activation. Initially, side scatter area versus forward scatter area density plots (SSC-A vs FSC-A) was used to identify the cell population of interest (inside the gate) and exclude the cellular debris. Subsequently, cellular doublets were excluded from analysis by forward scatter height (FSC-H) versus forward scatter width (FSC-W), followed by the side scatter height (FSC-H) versus side scatter width (FSC-W) parameters. The cells of interest were always inside the gate. Dendritic cells were separated from splenic single cells after cells gated according to cell type specific markers, being the CD11c⁺ and MCH-II⁺ molecules expression. Dendritic cells activation was evaluated by the expression the CD86⁺ co-stimulatory molecule. (b) The gate strategy of J774 cells. Cellular debris and cellular doublets were excluded as describe above. Murine J774 cells activation was evaluated after single cells gated by the expression of CD86⁺. CysA2 was able to induce CD89 upregulation on (c) dendritic cells and (d) J774 macrophages. (e) By SDS-PAGE followed by western-blotting analysis, we observed that CysA2 is probably binding to U937 human macrophages, as well as could also be phagocytized by them. (A-D) The analysis was performed in LSR Fortessa flow cytometers (BD Bioscience), and data were analyzed by FlowJo software (version 10.0.7 - Tree Star). All experiments were performed twice, in triplicate. MFI - median of fluorescence intensity; Mɸ - macrophages. *p < 0.05; ***p < 0.001.