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, research programs aiming at generating new anti-tumor compounds or at screening cDNA libraries. Stich approaches need to rely on reproducible, easy-to-handle, and rapid microplate-based cytotoxicity assays that are an-tenable to high-throughput screening (HTS) technologies. We describe it method for the direct measurement of cell death, based on the detection of a decrease in fluorescence observed following death induction in cells expressing enhanced green fluorescent protein (EGFP). Methods: Cell death was induced by it variety of apoptotic stimuli in various 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 fluorescence microplate reader. Results: Cells expressing EGFP exhibited a decrease in fluorescence when treated 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 fluorescence is directly quantifiable in a fluorescence-based microplate assay. Furthermore, analysis of EGFP protein content in cells undergoing cell death demonstrates that the decrease in fluorescence does not arise from degradation 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 for cytotoxicity evaluation. (C) 2001 Wiley Liss, Inc.