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. 2018 Jan 16;8:853. doi: 10.1038/s41598-017-19079-y

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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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Effect of cell culture insert pore density on cell differentiation. BBEC cultures were grown for 21 days at an ALI on membranes with pore densities of 2.0 × 10⁶ or 1.0 × 10⁸ pores/cm² before fixation. The BBEC cultures were subsequently immunostained to assess (A) ciliation (cilia - green; F-actin - red; nuclei - blue), (B) mucus production (mucus - green; cilia - red; nuclei - blue) and (C) tight-junction formation (tight-junctions - green; nuclei - blue) or (D) examined by SEM. Representative images are shown of BBECs grown on inserts with (i) 2.0 × 10⁶ and (ii) 1.0 × 10⁸ pores/cm². Quantitative analysis of ciliation of the apical surface of BBEC cultures was performed using (E) fluorescence intensity thresholding of immunostained cultures and (F) by counting the number of ciliated cells per field of view in H&E-stained sections as described in Fig. 2. Statistical significance was tested using an Ordinary one-way ANOVA: *** = P < 0.001; **** = P < 0.0001.