Detection of a decrease in green fluorescent protein fluorescence for the monitoring of cell death: An assay amenable to high-throughput screening technologies

Background: Reliable assessment of cell death is now pivotal to many, resea rch programs aiming at generating new anti-tumor compounds or at screening cDNA libraries. Stich approaches need to rely on reproducible, easy-to-hand le, and rapid microplate-based cytotoxicity assays that are an-tenable to h igh-throughput screening (HTS) technologies. We describe it method for the direct measurement of cell death, based on the detection of a decrease in f luorescence observed following death induction in cells expressing enhanced green fluorescent protein (EGFP). Methods: Cell death was induced by it variety of apoptotic stimuli in vario us EGFP-expressing mammalian cell lines, including those routinely used in anti-cancer drug screening. Decrease in fluorescence was assessed either by flow cytometry (and compared with other apoptotic markers) or by a fluores cence microplate reader. Results: Cells expressing EGFP exhibited a decrease in fluorescence when tr eated by various agents, such as chemotherapeutic drugs, UV irradiation, or caspase-independent cell death inducers. Kinetics and sensitivity of this EGFP-based assay were comparable to those of traditional apoptosis markers such as annexin-V binding, pro. gen species pidium iodide incorporation, or reactive oxygen production. We also show that the decrease in EGFP fluores cence is directly quantifiable in a fluorescence-based microplate assay. Fu rthermore, analysis of EGFP protein content in cells undergoing cell death demonstrates that the decrease in fluorescence does not arise from degradat ion of the protein. Conclusions: This novel GFP-based microplate assay combines sensitivity and rapidity, is easily amenable to HTS Setups, making it an assay of choice f or cytotoxicity evaluation. (C) 2001 Wiley Liss, Inc.