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Time-Lapse Imaging of Inflammasome-Dependent Cell Death and Extrusion in Enteroid-Derived Intestinal Epithelial Monolayers

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Pyroptosis

Part of the book series: Methods in Molecular Biology ((MIMB,volume 2641))

Abstract

Inflammasome-induced cell death is an epithelium-intrinsic innate immune response to pathogenic onslaught on epithelial barriers, caused by invasive microbes such as Salmonella Typhimurium (S.Tm). Pattern recognition receptors detect pathogen- or damage-associated ligands and elicit inflammasome formation. This ultimately restricts bacterial loads within the epithelium, limits breaching of the barrier, and prevents detrimental inflammatory tissue damage. Pathogen restriction is mediated via the specific extrusion of dying intestinal epithelial cells (IECs) from the epithelial tissue, accompanied by membrane permeabilization at some stage of the process. These inflammasome-dependent mechanisms can be studied in real time in intestinal epithelial organoids (enteroids), which allow imaging at high temporal and spatial resolution in a stable focal plane when seeded as 2D monolayers. The protocols described here involve the establishment of murine and human enteroid-derived monolayers, as well as time-lapse imaging of IEC extrusion and membrane permeabilization following inflammasome activation by S.Tm infection. The protocols can be adapted to also study other pathogenic insults or combined with genetic and pharmacological manipulation of the involved pathways.

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Authors and Affiliations

  1. Science for Life Laboratory, Department of Medical Biochemistry and Microbiology, Uppsala University, Uppsala, Sweden

    Petra Geiser, Jorik M. van Rijn & Mikael E. Sellin

  2. Current address: Princess Máxima Center for Pediatric Oncology, Utrecht, The Netherlands

    Jorik M. van Rijn

Authors
  1. Petra Geiser
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  2. Jorik M. van Rijn
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  3. Mikael E. Sellin
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Corresponding author

Correspondence to Mikael E. Sellin .

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Editors and Affiliations

  1. Department of Laboratory Medicine and Pathology, University of Washington, Seattle, WA, USA

    Susan L. Fink

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© 2023 The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature

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Geiser, P., van Rijn, J.M., Sellin, M.E. (2023). Time-Lapse Imaging of Inflammasome-Dependent Cell Death and Extrusion in Enteroid-Derived Intestinal Epithelial Monolayers. In: Fink, S.L. (eds) Pyroptosis. Methods in Molecular Biology, vol 2641. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-3040-2_17

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  • DOI: https://doi.org/10.1007/978-1-0716-3040-2_17

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  • Publisher Name: Humana, New York, NY

  • Print ISBN: 978-1-0716-3039-6

  • Online ISBN: 978-1-0716-3040-2

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