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. 2019 Nov 19;9:17045. doi: 10.1038/s41598-019-52624-5

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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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Demonstration of the gating strategy for the flow cytometric analysis of mouse CD4 CD25+Foxp3+ Treg (a), CD8+CD28− T cell (b), CD19+CD5+CD1d^hi Breg (c) from spleen. In this experiment a single cell suspension was prepared from the spleen of each group and stained with CD4 (FITC), CD25 (APC), Foxp3 (PE), CD8 (PE), CD28 (APC), CD19 (FITC), CD5 (APC), CD1d (PE) based on surface and intracellular staining protocols, respectively. Data were collected with FACSDiva flow cytometer and analyzed. Lymphocytes are identified by their scatter properties (FSC-A × SSC-A plot).