title: In vitro measurement of cell death with the annexin A5 affinity assay.
abstract: 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.
authors.affiliation: Department of Biochemistry, Cardiovascular Research Institute Maastricht, University of Maastricht, PO Box 616, 6200 MD, Maastricht, The Netherlands.
authors.forename: Hugo
authors.initials: H
authors.lastname: van Genderen
authors.forename: Heidi
authors.initials: H
authors.lastname: Kenis
authors.forename: Petra
authors.initials: P
authors.lastname: Lux
authors.forename: Lisette
authors.initials: L
authors.lastname: Ungeth
authors.forename: Cecile
authors.initials: C
authors.lastname: Maassen
authors.forename: Niko
authors.initials: N
authors.lastname: Deckers
authors.forename: Jagat
authors.initials: J
authors.lastname: Narula
authors.forename: Leo
authors.initials: L
authors.lastname: Hofstra
authors.forename: Chris
authors.initials: C
authors.lastname: Reutelingsperger
mesh_terms: 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
chemical_list: D017304:Annexin A5; D015415:Biomarkers; D005456:Fluorescent Dyes; D010718:Phosphatidylserines; D011419:Propidium