Green fluorescent protein as a novel tool to measure apoptosis and necrosis.
BACKGROUND
Several apoptosis-detecting methods are currently available. Many of them are work intensive and require the additional use of antibodies, dyes, specific substrates, or enzymatic reactions. A simple, fast, and reliable method was developed to test for apoptosis or necrosis using mouse and human cell lines (e.g., Jurkat, A20.2J, and PB3c cells) stably transfected with a vector coding for green fluorescent protein (GFP) as indicator cells.


METHODS
Apoptosis in GFP-transfected cell lines was induced either by soluble Fas-Ligand (sFasL), recombinant human TRAIL (rhTRAIL), or interleukin-3 (IL-3) deprivation. Necrosis was induced by polyclonal anti-A20 and complement treatment of GFP-transfected A20. Cells were analyzed by flow cytometry for GFP fluorescence. Propidium iodide and Annexin V staining were used to confirm the results obtained with the GFP-method.


RESULTS
Live GFP-transfected cells show a strong fluorescence intensity, which is significantly diminished upon induction of apoptosis, whereas necrotic GFP-transfected cells almost completely lose their GFP-associated fluorescence. Apoptosis but not necrosis of GFP-transfected cells was blocked by the use of a caspase inhibitor. The results are highly comparable to conventional apoptosis-detecting methods.


CONCLUSIONS
The advantage of our GFP-based assay compared with other methods is the analysis of apoptosis or necrosis without the necessity for additional staining or washing steps, making it an ideal tool for screening apoptotic or necrotic stimuli.
Strebel
A
A
Institute for Medical Microbiology, University of Basel, Basel, Switzerland.
Harr
T
T
Bachmann
F
F
Wernli
M
M
Erb
P
P
D000818:Animals; D017209:Apoptosis / Q000235:genetics; D051017:Apoptosis Regulatory Proteins; D002460:Cell Line / Q000473:pathology; D053222:Fas Ligand Protein; D005434:Flow Cytometry / Q000379:methods*; D049452:Green Fluorescent Proteins; D006801:Humans; D007202:Indicators and Reagents; D007377:Interleukin-3 / Q000378:metabolism; D019169:Jurkat Cells / Q000473:pathology; D008024:Ligands; D008164:Luminescent Proteins / Q000032:analysis* / Q000235:genetics; D008562:Membrane Glycoproteins / Q000502:physiology; D051379:Mice; D008807:Mice, Inbred BALB C; D008811:Mice, Inbred DBA; D009336:Necrosis; D012995:Solubility; D053221:TNF-Related Apoptosis-Inducing Ligand; D014162:Transfection; D014407:Tumor Cells, Cultured / Q000473:pathology*; D014409:Tumor Necrosis Factor-alpha / Q000502:physiology; D019014:fas Receptor / Q000378:metabolism
D051017:Apoptosis Regulatory Proteins; C505661:FASLG protein, human; D053222:Fas Ligand Protein; C505658:Fasl protein, mouse; D007202:Indicators and Reagents; D007377:Interleukin-3; D008024:Ligands; D008164:Luminescent Proteins; D008562:Membrane Glycoproteins; D053221:TNF-Related Apoptosis-Inducing Ligand; C505653:TNFSF10 protein, human; C505654:Tnfsf10 protein, mouse; D014409:Tumor Necrosis Factor-alpha; D019014:fas Receptor; D049452:Green Fluorescent Proteins