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. 2017 Jul 21;7:6163. doi: 10.1038/s41598-017-06369-8

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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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Goblet cell enrichment and airlifting culture of conjunctival epithelial cells (CECs). (A,B) Using Percoll density gradient to increase the initial goblet cell percentage in primary conjunctival epithelial cell populations. (A) Gene expression (RT-qPCR; fold change is expressed as 2^−ΔΔCT) of CECs before (control) and after goblet cell enrichment. CECs were separated into two populations after Percoll gradient separation – top and bottom (enriched). (B) PAS staining and immunohistochemistry (IHC) for CK4 and MUC5AC comparing the phenotypes of CECs with or without goblet cell enrichment. Scale bar 200 μm. (C) The schematics for airlifting (AL) and submerged (SM) cultures of CECs based on Transwell insert. H&E staining (D) and RT-qPCR (E) comparing CECs under AL and SM culture. Scale bar 100 μm. (F) IHC (top: CK4; bottom: MUC5AC) showing the difference in cell phenotypes between AL and SM cultures. Scale bar 50 μm (G) Mucin secretion (stained by Texas Red labeled dextran) visualized by confocal microscope (z scan). Both side view and 3D views are shown. Scale bar 100 μm. *p < 0.05; **p < 0.001.