In vitro measurement of cell death with the annexin A5 affinity assay.
One of the hallmarks of cell death is the cell surface-expression of phosphatidylserine. Expression of phosphatidylserine at the cell surface can be measured in vitro with the phosphatidylserine-binding protein annexin A5 conjugated to fluorochromes. This measurement can be made by flow cytometry or by confocal scanning-laser microscopy. The annexin A5 affinity assay comprises the incubation of cells stimulated to execute cell death with fluorescence-labeled annexin A5 and propidium iodide. Living cells are annexin A5-negative and propidium iodide negative, cells in the early phases of cell death are annexin A5 positive-and propidium iodide-negative, and secondary necrotic cells are annexin A5-positive and propidium iodide-positive. The entire procedure takes about 30 minutes for flow cytometry and 45 minutes for confocal scanning-laser microscopy. Various precautions and considerations are discussed further in the protocol described here.
van Genderen
Hugo
H
Department of Biochemistry, Cardiovascular Research Institute Maastricht, University of Maastricht, PO Box 616, 6200 MD, Maastricht, The Netherlands.
Kenis
Heidi
H
Lux
Petra
P
Ungeth
Lisette
L
Maassen
Cecile
C
Deckers
Niko
N
Narula
Jagat
J
Hofstra
Leo
L
Reutelingsperger
Chris
C
D017304:Annexin A5 / Q000032:analysis* / Q000737:chemistry; D017209:Apoptosis; D015415:Biomarkers / Q000032:analysis; D005434:Flow Cytometry; D005456:Fluorescent Dyes / Q000032:analysis / Q000737:chemistry; D006801:Humans; D019169:Jurkat Cells; D018613:Microscopy, Confocal; D010718:Phosphatidylserines / Q000032:analysis* / Q000378:metabolism; D011419:Propidium / Q000032:analysis
D017304:Annexin A5; D015415:Biomarkers; D005456:Fluorescent Dyes; D010718:Phosphatidylserines; D011419:Propidium