Name: Video: Assessing Human Colonoid Cell Death Using SYTOX Green Fluorescence Imaging
Uploaded: 2024-08-02T13:50:00
Duration: 4 min 17 s
Description: 208 Views. Assessing Human Colonoid Cell Death Using SYTOX Green Fluorescence Imaging

assessing human colonoid cell death using sytox green fluorescence imaging

Quantifying Cell Death in Human Colonoids in Response to Cytotoxic Perturbagens

The intestinal epithelium creates a physical semipermeable barrier between the contents of the gut lumen and the underlying tissues. To maintain this barrier effectively, intestinal epithelial cells (IECs) undergo an extremely high cellular turnover, with a continuous cycle of cell death and regeneration. However, during inflammatory disorders, such as inflammatory bowel disease (IBD), higher levels of aberrant cell death occur1. This may promote a breakdown in barrier function and activation of the immune system, triggering further inflammation. In Crohn&#39;s disease (CD), a form of IBD, it has been shown that cytokine signaling contributes to the increased levels of IEC death2. By studying how cytokine signaling induces cell death of IECs, it is hoped that improved treatments can be developed for patients with IBD and other intestinal inflammatory disorders1.
In biology and drug target discovery research, synergy is generally understood to occur when a biological system treated with combinations of individual stimuli shows a response to the combination that is greater than the combined additive effects of the single stimuli alone. Synergistic interactions between cytokines have been well documented in driving innate antiviral responses3. Cytokines have also been known to induce cell death synergistically, including in IECs4. However, the role synergistic cytotoxic cytokine signaling plays in intestinal inflammatory disorders such as IBD is understudied.
Human intestinal organoids are 3D microtissues produced in vitro that are generated from intestinal epithelial stem cells. Intestinal organoids can be grown from gut mucosal biopsies obtained from patients with IBD and retain many characteristics of the disease5,6. Organoids have proven to be an ideal model system for studying cytokine cytotoxicity in the context of intestinal inflammation7,8. Previously, our group has characterized the synergistic killing effects of the IBD-relevant cytokines IFN-&#947; and TNF-&#945; in CD patient-derived colonic organoids (colonoids)9,10. However, the exact mechanisms involved in mediating this form of synergistic cell death remain elusive. There are also potentially many more uncharacterized cytotoxic cytokine interactions that are relevant to intestinal inflammatory disorders.
Several protocols are available to study cell death in intestinal organoids10,11,12,13; however, they each have drawbacks. Some of these techniques only measure cell viability and do not measure cell death directly, are incapable of evaluating single-organoid responses, or require expensive equipment and complex protocols. Robust and straightforward methodologies are needed to quantify organoid cell death and perturbagen interactions in intestinal organoids. The protocol we present is a simple and inexpensive approach to measure single-organoid responses to cytotoxic cytokines but can be used for any type of stimulus or perturbagen. We also demonstrate how to use the Bliss independence synergy model to calculate a coefficient of perturbagen interaction (CPI) that describes cytotoxic cytokine interactions.

Author Spotlight: Understanding Cytokine-Induced Cell Death in Intestinal Epithelial Cells Using Human Organoids

Quantification of Cytokine-Induced Cell Death in Human Colonic Organoids Using Live Fluorescence Microscopy

Assessing Human Colonoid Cell Death Using SYTOX Green Fluorescence Imaging

assessing-human-colonoid-cell-death-using-sytox-green-fluorescence

Research

JoVE Journal

Biology

208 Views. Assessing Human Colonoid Cell Death Using SYTOX Green Fluorescence Imaging

Video: Assessing Human Colonoid Cell Death Using SYTOX Green Fluorescence Imaging

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-&#945; and IFN-&#947;, 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.

This protocol describes a simple and cost-effective method to investigate and quantify cell death in human colonic organoids in response to cytotoxic perturbagens such as cytokines. The approach employs a fluorescent cell death dye (SYTOX Green Nucleic Acid Stain), live fluorescence microscopy, and open-source image analysis software to quantify single-organoid responses to cytotoxic stimuli.

Intestinal epithelial cell (IEC) death is increased in patients with inflammatory bowel diseases (IBD) such as ulcerative colitis (UC) and Crohn's disease ...