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 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.