PMID- 38314824
OWN - NLM
STAT- MEDLINE
DCOM- 20240207
LR  - 20240423
IS  - 1940-087X (Electronic)
IS  - 1940-087X (Linking)
IP  - 203
DP  - 2024 Jan 19
TI  - Live Calcium Imaging of Virus-Infected Human Intestinal Organoid Monolayers Using 
      Genetically Encoded Calcium Indicators.
LID - 10.3791/66132 [doi]
AB  - 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.
FAU - Gebert, J Thomas
AU  - Gebert JT
AD  - Alkek Center for Metagenomics and Microbiome Research, Department of Molecular 
      Virology and Microbiology, Baylor College of Medicine.
FAU - Scribano, Francesca J
AU  - Scribano FJ
AD  - Alkek Center for Metagenomics and Microbiome Research, Department of Molecular 
      Virology and Microbiology, Baylor College of Medicine.
FAU - Engevik, Kristen A
AU  - Engevik KA
AD  - Alkek Center for Metagenomics and Microbiome Research, Department of Molecular 
      Virology and Microbiology, Baylor College of Medicine.
FAU - Hyser, Joseph M
AU  - Hyser JM
AD  - Alkek Center for Metagenomics and Microbiome Research, Department of Molecular 
      Virology and Microbiology, Baylor College of Medicine; joseph.hyser@bcm.edu.
LA  - eng
GR  - F31 AI169983/AI/NIAID NIH HHS/United States
GR  - F31 DK132942/DK/NIDDK NIH HHS/United States
GR  - F30 DK131828/DK/NIDDK NIH HHS/United States
GR  - R01 DK115507/DK/NIDDK NIH HHS/United States
GR  - R01 AI158683/AI/NIAID NIH HHS/United States
PT  - Journal Article
PT  - Video-Audio Media
DEP - 20240119
PL  - United States
TA  - J Vis Exp
JT  - Journal of visualized experiments : JoVE
JID - 101313252
RN  - SY7Q814VUP (Calcium)
SB  - IM
MH  - Humans
MH  - *Calcium/analysis
MH  - *Intestines
MH  - Intestinal Mucosa/chemistry
MH  - Organoids/chemistry
MH  - Microscopy, Fluorescence/methods
EDAT- 2024/02/05 14:45
MHDA- 2024/02/07 06:42
CRDT- 2024/02/05 08:48
PHST- 2024/02/07 06:42 [medline]
PHST- 2024/02/05 14:45 [pubmed]
PHST- 2024/02/05 08:48 [entrez]
AID - 10.3791/66132 [doi]
PST - epublish
SO  - J Vis Exp. 2024 Jan 19;(203). doi: 10.3791/66132.