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Establishment of Intestinal Epithelial Cell Monolayers and Their Use in Calcium Switch Assay for Assessment of Intestinal Tight Junction Assembly

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Permeability Barrier

Part of the book series: Methods in Molecular Biology ((MIMB,volume 2367))

Abstract

Intestinal barrier function relies primarily on the assembly and integrity of tight junctions, which forms a size-selective barrier. This barrier restricts paracellular movement of solutes in various types of epithelia. Of note, extracellular Ca2+ concentration affects tight junction assembly. Therefore, the removal and re-addition of Ca2+ into cell culture medium of cultured intestinal epithelial cells causes destabilization and reassembly of tight junction to membrane periphery near apical surface, respectively. Based on this principle, the Ca2+-switch assay was established to investigate tight junction assembly in fully differentiated intestinal epithelial cells. This chapter provides a stepwise protocol for culture of intestinal epithelial cell monolayers using T84 cell line as an in vitro model and the Ca2+-switch assay for evaluating tight junction assembly.

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Acknowledgments

This research project is funded by Chulabhorn Royal Academy (to PP), by grant DBG6180029 from Thailand Science Research and Innovation and National Research Council of Thailand (to CM), and by German Research Foundation (DFG, STE 2735/2-1) (to PRS).

Author Contributions:

Pawin Pongkorpsakol: Conceptualization; data curation; formal analysis; investigation; methodology; supervision; validation; writing-original draft; Writing-review and editing.

Wilasinee Satianrapapong: Conceptualization; Data curation; formal analysis; investigation; methodology; Writing-original draft.

Preedajit Wongkrasant: Investigation; methodology; formal analysis

Peter R. Steinhagen: Writing-original draft; Writing-review and editing.

Nuttha Tuangkijkul: Investigation; formal analysis

Nutthapoom Pathomthongtaweechai: Writing-review and editing.

Chatchai Muanprasat: Supervision; Providing facilities and all supports for experiments.

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Authors and Affiliations

  1. Faculty of Medicine and Public Health, HRH Princess Chulabhorn College of Medical Science, Chulabhorn Royal Academy, Bangkok, Thailand

    Pawin Pongkorpsakol

  2. Section for Translational Medicine, Faculty of Medicine Ramathibodi Hospital, Mahidol University, Bangkok, Thailand

    Wilasinee Satianrapapong

  3. Department of Physiology, Faculty of Science, Mahidol University, Bangkok, Thailand

    Preedajit Wongkrasant

  4. Department of Hepatology and Gastroenterology, Charité Medical School, Berlin, Germany

    Peter R. Steinhagen

  5. Faculty of Medicine Ramathibodi Hospital, Mahidol University, Bangkok, Thailand

    Nuttha Tuangkijkul

  6. Chakri Naruebodindra Medical Institute, Faculty of Medicine, Ramathibodi Hospital, Mahidol University, Samut Prakan, Thailand

    Nutthapoom Pathomthongtaweechai & Chatchai Muanprasat

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  1. Pawin Pongkorpsakol
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  2. Wilasinee Satianrapapong
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Correspondence to Pawin Pongkorpsakol .

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  1. Ottawa, ON, Canada

    Kursad Turksen

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Pongkorpsakol, P. et al. (2021). Establishment of Intestinal Epithelial Cell Monolayers and Their Use in Calcium Switch Assay for Assessment of Intestinal Tight Junction Assembly. In: Turksen, K. (eds) Permeability Barrier. Methods in Molecular Biology, vol 2367. Humana, New York, NY. https://doi.org/10.1007/7651_2021_347

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  • DOI: https://doi.org/10.1007/7651_2021_347

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  • Publisher Name: Humana, New York, NY

  • Print ISBN: 978-1-0716-1672-7

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