Language
English
Publication Date
1-19-2024
Journal
Journal of Visualized Experiments (JoVE)
DOI
10.3791/66132
PMID
38314824
PMCID
PMC11157669
PubMedCentral® Posted Date
1-18-2025
PubMedCentral® Full Text Version
Author MSS
Abstract
Calcium signaling is an integral regulator of nearly every tissue. Within the intestinal epithelium, calcium is involved in the regulation of secretory activity, actin dynamics, inflammatory responses, stem cell proliferation, and many other uncharacterized cellular functions. As such, mapping calcium signaling dynamics within the intestinal epithelium can provide insight into homeostatic cellular processes and unveil unique responses to various stimuli. Human intestinal organoids (HIOs) are a high-throughput, human-derived model to study the intestinal epithelium and thus represent a useful system to investigate calcium dynamics. This paper describes a protocol to stably transduce HIOs with genetically encoded calcium indicators (GECIs), perform live fluorescence microscopy, and analyze imaging data to meaningfully characterize calcium signals. As a representative example, 3-dimensional HIOs were transduced with lentivirus to stably express GCaMP6s, a green fluorescent protein-based cytosolic GECI. The engineered HIOs were then dispersed into a single-cell suspension and seeded as monolayers. After differentiation, the HIO monolayers were infected with rotavirus and/or treated with drugs known to stimulate a calcium response. An epifluorescence microscope fitted with a temperature-controlled, humidified live-imaging chamber allowed for long-term imaging of infected or drug-treated monolayers. Following imaging, acquired images were analyzed using the freely available analysis software, ImageJ. Overall, this work establishes an adaptable pipeline for characterizing cellular signaling in HIOs.
Keywords
Humans, Calcium, Intestines, Intestinal Mucosa, Organoids, Microscopy, Fluorescence
Published Open-Access
yes
Recommended Citation
Gebert, J Thomas; Scribano, Francesca J; Engevik, Kristen A; et al., "Live Calcium Imaging of Virus-Infected Human Intestinal Organoid Monolayers Using Genetically Encoded Calcium Indicators" (2024). Faculty, Staff and Students Publications. 6196.
https://digitalcommons.library.tmc.edu/baylor_docs/6196