High-throughput technology: green fluorescent protein to monitor cell death
Marylène Fortin1, Ann-Muriel Steff, Patrice Hugo
1Topigen Pharmaceuticals, Montreal, Quebec, Canada.
Abstract:
Reliable assessment of cell death is now pivotal to many research programs aiming at generating new antitumor compounds or at screening cDNA libraries to identify genes with pro- or antiapoptotic functions. Such approaches need to rely on reproducible, easy handling, and rapid microplate-based cytotoxicity assays that are amenable to high-throughput screening technologies. We describe here a method for the direct measurement of cell death, based on the detection of a decrease in fluorescence observed following death induction in cells stably expressing enhanced green fluorescent protein (EGFP). Our data clearly show that such a decrease in EGFP fluorescence after cell death induction happens in various cell types, including those routinely used in anticancer drug screening (i.e., murine and human, lymphoid, fibroblastic, or epithelial cell lines). Moreover, the decrease in EGFP fluorescence is observed in cells induced to die by a variety of apoptosis-inducing agents, such as glucocorticoids (dexamethasone), DNA- damaging agents (etoposide, cisplatin), microtubule disorganizers (paclitaxel), protein kinase C inhibitors (staurosporine), or a caspase-independent apoptotic stimulus (CD45 crosslinking). A decrease in fluorescence can be assessed either by flow cytometry or with a fluorescence microplate reader. The kinetics and specificity of this EGFP-based assay were comparable with those of other conventional techniques used to detect cell death. This novel EGFP-based microplate assay combines sensitivity and rapidity and is amenable to high-throughput setups, making it an assay of choice for evaluation of cell cytotoxicity.
Insights
A new assay directly measures cell death by detecting decreased fluorescence in cells expressing enhanced green fluorescent protein (EGFP). This rapid, high-throughput method is ideal for screening anticancer compounds and identifying apoptosis-related genes.
Area of Science:
- Biotechnology
- Cell Biology
- Drug Discovery
Background:
- Accurate cell death assessment is crucial for developing new anticancer drugs.
- Existing cytotoxicity assays often lack the speed and throughput required for high-throughput screening.
Purpose of the Study:
- To develop a direct, rapid, and high-throughput microplate-based assay for measuring cell death.
- To utilize enhanced green fluorescent protein (EGFP) expression for detecting cell death.
Main Methods:
- Cells stably expressing EGFP were induced to undergo cell death.
- A decrease in EGFP fluorescence was measured using flow cytometry and a fluorescence microplate reader.
- The assay was validated across various cell types and apoptosis-inducing agents.
Main Results:
- A consistent decrease in EGFP fluorescence was observed upon induction of cell death in diverse cell lines.
- The EGFP-based assay demonstrated comparable kinetics and specificity to conventional cell death detection methods.
- The assay proved effective with various apoptosis-inducing agents, including chemical and physical stimuli.
Conclusions:
- The novel EGFP-based microplate assay offers a sensitive, rapid, and reproducible method for direct cell death measurement.
- This assay is suitable for high-throughput screening applications in anticancer drug discovery and functional genomics.
- The assay provides a valuable tool for evaluating cytotoxicity across various experimental setups.
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