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.