Protocol for the application of single-cell damage in murine intestinal organoid models

Spatially defined organoid damage enables the study of cellular repair processes. However, capturing dynamic events in living tissues is technically challenging. Here, we present a protocol for the application of single-cell damage in intestinal organoid models. We describe steps for isolating and c...

Full description

Saved in:
Bibliographic Details
Published in:STAR protocols 2024-09, Vol.5 (3), p.103153, Article 103153
Main Authors: Seidler, Anna Elisabeth, Donath, Sören, Gentemann, Lara, Buettner, Manuela, Heisterkamp, Alexander, Kalies, Stefan
Format: Article
Language:English
Subjects:
Biophysics
Microscopy
Molecular Biology
Organoids
Single Cell
Citations: Items that this one cites
Online Access:Get full text
Tags: Add Tag
No Tags, Be the first to tag this record!
Description
Summary:Spatially defined organoid damage enables the study of cellular repair processes. However, capturing dynamic events in living tissues is technically challenging. Here, we present a protocol for the application of single-cell damage in intestinal organoid models. We describe steps for isolating and cultivating murine colon organoids, lentivirus generation and transduction of organoids, single-cell ablation by a femtosecond laser, and follow-up imaging analysis. We provide examples for the image acquisition pipeline of dynamic processes in organoids using a confocal microscope. For complete details on the use and execution of this protocol, please refer to Donath et al.1,2 [Display omitted] •Instructions for the isolation, cultivation, and transduction of intestinal organoids•Guidelines for the design of lentiviral transfer plasmids suitable for your research•Precise single-cell ablation in 3D organoids with a femtosecond laser•Confocal imaging of dynamic wound healing processes in intestinal organoids Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics. Spatially defined organoid damage enables the study of cellular repair processes. However, capturing dynamic events in living tissues is technically challenging. Here, we present a protocol for the application of single-cell damage in intestinal organoid models. We describe steps for isolating and cultivating murine colon organoids, lentivirus generation and transduction of organoids, single-cell ablation by a femtosecond laser, and follow-up imaging analysis. We provide examples for the image acquisition pipeline of dynamic processes in organoids using a confocal microscope.
ISSN:2666-1667
2666-1667
DOI:10.1016/j.xpro.2024.103153