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. 2018 Dec 5;8:17651. doi: 10.1038/s41598-018-36121-9

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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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Characterization of basal signaling patterns in freshly isolated and cryopreserved primary B cells. B cells were isolated from healthy blood donors and analyzed immediately (n = 10; fresh) or after cryopreservation (n = 10; cryo). The cells were fixed, permeabilized and stained with anti-CD19 surface marker and phospho-specific antibodies as described in Methods. The fluorescence signals were detected by flow cytometry and analyzed in Cytobank. The basal fluorescence signals were measured relative to IgGκ isotype control and shown as arcsinh ratio (box-and-whisker; outliers are indicated separately). Statistical significance was calculated by a paired t-test with correction for multiple comparisons using the Holm-Sidak’s method, ****p < 0.00001. The plots to the right show raw flow cytometry data from one representative donor (fresh). Each plot corresponds to the protein shown in the aligned box-and-whisker plot, with phospho-protein (x-axis) plotted against FSC-A (y-axis).