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. 2018 Feb 23;8:3565. doi: 10.1038/s41598-018-21895-9

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© The Author(s) 2018

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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Expression of B cell markers genes in IgM⁺ B cells treated with CpG and/or condition supernatants. IgM⁺ B cells from peripheral blood (PB), head kidney (HK) and spleen leukocytes were MACS sorted and analyzed by RT-qPCR for expression of (a) secreted IgM (sigm) and (b) membrane IgM (migm). Gene expression data at each time point were normalized against the reference gene EF1aB and fold changes were calculated using the unstimulated sample at the same time point⁷⁴. Data represent mean ± s.e.m. from at least three individuals (n = 3 to 6). The line intersecting the y-axis at 1 represents the unstimulated control that the fold change of the treatments are in relation to. Significant fold changes (p < 0.05) are indicated by *. CAS; Control adherent cell supernatant, PAS; Pulse adherent cell supernatant.