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. 2019 Jul 26;8(4):653–655.e5. doi: 10.1016/j.jcmgh.2019.07.009

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© 2019 The Authors

This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

PMC Copyright notice

VIP-assisted ALI culture generates physiologic mucus layer above intestinal epithelial cells. (A) Culture schematic. (B) Dose-dependent water secretion of VIP at 24 hours. (C) Immunofluorescence of monolayers differentiated under submerged and ALI conditions in the presence of VIP. Green: anti-Muc2; blue: Hoechst 33342. (D) Removal of hydrated mucus with forceps. (E) Side-view confocal micrograph showing tissues with mucus separating bacteria from epithelial cells at 0 and 6 days, respectively. Green: green fluorescent protein (GFP)–expressing Escherichia coli; blue: Hoechst 33342. (F) Plot of mucus thickness vs duration of ALI. (G) Representative side-view confocal micrograph showing 1 μm red fluorescent beads separated from epithelial cells by mucus. (H) Immunofluorescence staining of paraffin-embedded, sectioned monolayers. DM, differentiation medium.