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. 2019 Nov 6;9:16115. doi: 10.1038/s41598-019-52562-2

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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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Maintenance of the IL-17A and IFN-γ phenotype of sorted and frozen-thawed cells upon subculture. (a) Sorted IL-17A+ cells stimulated with α-CD3/α-CD28 were cultured with IL-2 with or without the polarizing cytokines TGF-β1 and IL-6 for 9 days and analyzed by flow cytometry (ICS). Percentages of IL-17A+/IFN-γ+ double positive cells and of IL-17A-/IFN-g+ cells are shown. (b) Thawed IL-17A+ cells were cultured for 22 days and analyzed by ICS at days 12 and 22. Percentages of IL-17A+ cells (all: both IL-17A+/IFN-γ- and double positive cells) and IL-17A-/IFN-γ+ cells are shown. Results from two cows (C1 & C2) are shown.