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. Such approaches need to rely on reproducible, easy-to-handle, and rapid microplate-based cytotoxicity assays that are amenable to high-throughput screening (HTS) technologies. We describe a 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 a 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, propidium iodide incorporation, or reactive oxygen species 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.
Steff
A M
AM
PROCREA BioSciences, Division of Research and Development, Montreal, Quebec, Canada.
Fortin
M
M
Arguin
C
C
Hugo
P
P
D016475:3T3 Cells / Q000737:chemistry / Q000473:pathology*; D000818:Animals; D017209:Apoptosis; D015153:Blotting, Western; D003603:Cytotoxins / Q000633:toxicity; D004354:Drug Screening Assays, Antitumor / Q000379:methods*; D005434:Flow Cytometry / Q000379:methods; D049452:Green Fluorescent Proteins; D007202:Indicators and Reagents / Q000032:analysis*; D008164:Luminescent Proteins / Q000032:analysis*; D051379:Mice; D012680:Sensitivity and Specificity; D013050:Spectrometry, Fluorescence; D014407:Tumor Cells, Cultured / Q000737:chemistry / Q000473:pathology*
D003603:Cytotoxins; D007202:Indicators and Reagents; D008164:Luminescent Proteins; D049452:Green Fluorescent Proteins