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. 2017 Jul 19;7:5814. doi: 10.1038/s41598-017-06261-5

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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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Cytokine levels of CD4 T cells in immunized C57BL/6 mice. 4 days after the 3^rd vaccination, the splenocytes of immunized C57BL/6 mice were prepared. For cytokines level in CD4 T cells analysis, the splenocytes were stimulated with Cell Stimulation Cocktail (eBioscience) and treated with Protein Transport Inhibitor Cocktail (eBioscience) for cytokines production by flow cytometry. (A) The samples were intracellularly stained with anti-mouse CD4-FITC/IFN-γ-PE for Th1 cytokine analysis, anti-mouse CD4-FITC/IL-4-PE for Th2 cytokine analysis or anti-mouse CD4-FITC/IL-21-PE for Tfh cytokine analysis. The CD4⁺IFN-γ⁺ cells, CD4⁺IL-4⁺cells or CD4⁺IL-21⁺ cells were counted relatively to total CD4⁺ T cells. (B) The statistical results of Th1, Th2 and Tfh cytokines were shown. Shown in each panel is 1 of at least 3 experiments with similar results. Bar, mean and SD from 3 independent experiments, each using at least three mice per group (n = 3); *p < 0.05; **p < 0.01.