Real-time tracking of cell death in human intestinal organoids: A quantitative imaging approach

Organoids are self-renewing three-dimensional tissue models that can be derived from patient samples. Their recapitulation of the structure and function of native tissues makes them powerful tools for studying development, physiology, disease mechanisms and personalized medicine in vitro. However, quantitative assessment of cell death in organoid systems remains challenging due to their structural complexity and dynamic responses. Here, we present a robust pipeline combining real-time Incucyte® live-cell imaging with ImageJ-based quantitative analysis to measure cell death kinetics in human intestinal organoids. Organoids are cultured in the low-viscosity matrix suspension culture method, labelled with cell death-specific Incucyte® Cytotox Red Dye, and imaged via brightfield and fluorescence channels. A custom ImageJ analysis pipeline enables calculation of cell death overtime with high sensitivity and reproducibility. This approach overcomes the limitations of endpoint assays, delivering precise kinetic quantification of organoid cell death in response to cytokine stimulation, drug treatments, or genetic perturbations. The workflow is broadly applicable across diverse organoid systems and offers a scalable, standardized platform for interrogating cell death dynamics in basic and translational research.