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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.

Document Type

Article

Publication Date

2026

Notes/Citation Information

© 2026 Oza et al . This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Digital Object Identifier (DOI)

https://doi.org/10.1371/journal.pone.0343016

Funding Information

National Institutes of Health, Grant/ Award Numbers R37CA227656 (to J.S.B.) and F99CA294265 (to V.D.O.) [https://www.nih.gov/] and the Kentucky Pediatric Cancer Research Trust Fund PON27282400002665 [https://www.chfs.ky.gov/agencies/dph/dpqi/cdpb/Pages/pcrtf.aspx]. This research is also supported by the Flow Cytometry and Immune Monitoring Shared Resource of the Markey Cancer Center (P30CA177558) [https://ukhealthcare.uky.edu/markey-cancer-center/research/srf/flow]. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

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