Abstract
The Genetically Encoded Death Indicator (GEDI) is a ratiometric, dual-fluorescence biosensor that enables real-time detection of cell death through calcium influx. Originally developed for use in neurodegeneration models, GEDI can be applied to cancer cells to quantify therapy-induced death at single-cell resolution. This protocol details how to generate GEDI-expressing cancer cell lines, empirically determine stress-induced GEDI thresholds using radiation or chemotherapeutic agents, and perform time-resolved imaging and image analysis to track cell fate. This workflow is optimized for high-throughput drug and radiation screening in heterogeneous populations and is especially useful for identifying chemo- and radio-resistant subclones. Key limitations include the need for empirical GEDI threshold calibration for each treatment condition and careful standardization of imaging parameters. The protocol outputs include GEDI ratio values, single-cell time-of-death annotations, and whole-cell morphological data in parallel, which can be linked to downstream applications such as FACS-based isolation of live or dying subpopulations, transcriptomic profiling of resistant clones, or in vivo validation using xenografts or organotypic slice culture.