Quantification of Cytokine-Induced Cell Death in Human Colonic Organoids Using Live Fluorescence Microscopy
Abstract
Intestinal epithelial cell (IEC) death is increased in patients with inflammatory bowel diseases (IBD) such as ulcerative colitis (UC) and Crohn's disease (CD). This can contribute to defects in intestinal barrier function, exacerbation of inflammation, and disease immunopathogenesis. Cytokines and death receptor ligands are partially responsible for this increase in IEC death. IBD-relevant cytokines, such as TNF-α and IFN-γ, are cytotoxic to IECs both independently and in combination. This protocol describes a simple and practical assay to quantify cytokine-induced cytotoxicity in CD patient-derived colonic organoids using a fluorescent cell death dye (SYTOX Green Nucleic Acid Stain), live fluorescence microscopy, and open-source image analysis software. We also demonstrate how to use the Bliss independence mathematical model to calculate a coefficient of perturbagen interaction (CPI) based on organoid cytotoxicity. The CPI can be used to determine if interactions between cytokine combinations or other types of perturbagens are antagonistic, additive, or synergistic. This protocol can be implemented to investigate the cytotoxic activity of cytokines and other perturbagens using patient-derived colonic organoids.
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- Authors
- Flood, P., & Nally, K.
- Affiliations
- University College Cork (IE)
- ORCID
- Flood, Peter
- Venue
- Journal of Visualized Experiments
- Publisher
- MyJove Corporation
- Published
- 2024-08-02
- Issue
- 210
- ISSN
- 1940-087X (ISSN-L)
- OpenAlex
- W4401317126
- Semantic Scholar
- d06052ee785d3e0ea40e43ca12b1cc9703099f30
- Cited by
- 2
- References
- 28
- Citations by year
- 2026: 2
- FWCI
- 0.47
- Type
- journal article
- Language
- en
- Open access
- closed
- Locations
- Journal of Visualized Experiments (publishedVersion)
- PubMed (publishedVersion)
- Topics
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Genetics › Inflammatory Bowel Disease
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Cell death mechanisms and regulation
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Cancer Research › NF-κB Signaling Pathways
- Keywords
- cytokine-induced cell death, colonic organoids, intestinal epithelial cells, inflammatory bowel disease, ulcerative colitis, Crohn's disease, TNF-α, interferon-gamma, death receptor ligands, Bliss independence model, synergistic and antagonistic effects, patient-derived organoids
- MeSH terms
- Colon, Humans, Microscopy, Fluorescence, Organoids, Cytokines, Cell Death
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