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. 2017 Sep 14;7:11644. doi: 10.1038/s41598-017-11271-4

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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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Z-stack series of basolaterally added DCs and apically applied macrophages to analyse the distribution in the co-culture system without any stimulus. The distribution of monocyte-derived DCs (a) or macrophages (b) (2.5 × 10⁵ cells/Transwell) was analysed over time via CLSM. Tight junctions of epithelial cells were stained using actin (white), nuclei using Höchst (blue) and immune cells using CFSE (2.5 µM) prior to additon to epithelia. (a) DCs did not migrate from the basolateral side of the Transwell to the apical layers over a time course of 24 h (1h-3h-6h-24h) and as analysed by x-y projections with respective side views – an example is given at the lower right. (b) Apically added macrophages (green) did not show any signs of activation, e.g. clustering on the SAE layer (actin, white; nucleus, blue) even after 24 h as analysed by CLSM (upper) and SEM. Yellow arrows indicate macrophages on the apical side of the epithelium. For CLSM at least 3 different areas each containing ~25 cells per condition were captured.