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. 2021 Mar 25;19:83. doi: 10.1186/s12951-021-00830-7

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© The Author(s) 2021

Open AccessThis 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 licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence 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 licence, visit http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data.

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Fig. 2 — Surface marker changes and migration behavior of immune cell subsets after CCL25 stimulation. After stimulation with different dosages of CCL25 (10, 100, 750 nM), immunofluorescence staining with antibody panels and subsequent flow cytometry was performed to detect leukocyte activation and macrophage polarization based on surface marker expression. Additionally, a Boyden-Chamber migration assay was performed with a CCL25 dilution series (0.01–1000 nM). a HLA-DR expression levels of different immune cell subsets stimulated with 10, 100 and 750 nM CCL25 over three days are shown as the mean of the percentage of marker positive cells + SD normalized to the negative control (red dotted line); n = 3. b The migration of immune cell subsets towards a dilution series of CCL25 is displayed as the mean + SD of the number of migrated cells (n = 4). Macrophage polarization through CCL25 stimulation was analyzed by immunofluorescence antibody staining and flow cytometric analysis of M0 and M1 macrophages against either c M1 polarization markers CD80 and HLA-DR or d M2 polarization markers CD163 and CD206 (n = 4). Surface marker expression levels are shown as the mean + SD of normalized values compared to the negative control (red dotted line). Statistical significance was analyzed using either a student’s T-test or the Mann–Whitney-U test depending on whether data followed a normal distribution. Individual p values are given above the bars if lower than p = 0.20. ΜΦ Macrophages