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Nat Protoc. 2006;1(1):363-7.

In vitro measurement of cell death with the annexin A5 affinity assay.

Author information

1
Department of Biochemistry, Cardiovascular Research Institute Maastricht, University of Maastricht, PO Box 616, 6200 MD, Maastricht, The Netherlands.

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.

PMID:
17406257
DOI:
10.1038/nprot.2006.55
[Indexed for MEDLINE]

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