Assessment of Apoptosis (Programmed Cell Death) by Flow Cytometry

A useful feature of therapeutic antibodies is the ability to kill the cells to which they bind. Antibodies are capable of mediating cell killing in a variety of ways. Apoptosis, complement-mediated mechanisms, and antibody-dependent cellular cytotoxicity are all effects that can be assayed to characterize lead antibody candidates. Extensive, multidose characterizations of a series of candidates can be performed in a short amount of time using assays developed for high-throughput flow cytometry systems. Here, we describe a simple multiplexed flow assay performed using Annexin V and propidium iodide that measures an early marker of apoptosis. When cells enter apoptosis, phosphatidyl serine (PS), which is normally found on the inside of the cytoplasmic membrane, is found on the extracellular surface of the membrane, thus revealing Annexin V–binding sites. Because binding of Annexin V to PS is calcium dependent, the buffers used for this assay must contain 1 m m calcium. The calcium dependence can also be used to test whether the Annexin V staining is specific. Thus, if the staining is performed in the presence of 1 m m EDTA, binding of Annexin V should be inhibited. The addition of propidium iodide allows subsequent stages of apoptosis and eventual cell death to be distinguished. For flow cytometry, this assay is best performed on suspension cells.