Regulatory Status Legend

Description

Apoptosis is a normal physiologic process which occurs during embryonic development as well as in maintenence of tissue homeostasis. The apoptotic program is characterized by certain morphologic features, including loss of plasma membrane asymmetry and attachment, condensation of the cytoplasm and nucleus, and internucleosomal cleavage of DNA. Loss of plasma membrane is one of the earliest features. In apoptotic cells, the membrane phospholipid phosphatidylserine (PS) is translocated from the inner to the outer leaflet of the plasma membrane, thereby exposing PS to the external cellular environment. Annexin V is a 35-36 kDa Ca2+ dependent phospholipid-binding protein that has a high affinity for PS, and binds to cells with exposed PS. Annexin V may be conjugated to fluorochromes, such as fluorescein isothiocyanate (FITC), to serve as a sensitive probe for flow cytometric analysis of cells that are undergoing apoptosis. Since externalization of PS occurs in the earlier stages of apoptosis, Annexin V staining can identify apoptosis at an earlier stage than assays based on nuclear changes such as DNA fragmentation. In addition, Annexin V binding sites may be blocked by incubating cells with purified recombinant Annexin V prior to incubation with one of the fluorochrome labeled formats of Annexin V.

Annexin V staining precedes the loss of membrane integrity which accompanies the latest stages of cell death resulting from either apoptotic or necrotic processes. Therefore, staining with Annexin V is typically used in conjunction with a vital dye such as 7-Amino-Actinomycin (7-AAD) to allow the investigator to identify early apoptotic cells (7-AAD negative, Annexin V positive). Viable cells with intact membranes exclude 7-AAD, whereas the membranes of dead and damaged cells are permeable to 7-AAD. For example, cells that are considered viable are both Annexin V and 7-AAD negative while cells that are in early apoptosis are Annexin V positive and 7-AAD negative, while cells that are in late apoptosis or already dead are both Annexin V and 7-AAD positive. This assay does not distinguish between cells that have undergone apoptotic death versus those that have died as a result of a necrotic pathway because in either case, the dead cells will stain with both Annexin V and 7-AAD. However, when apoptosis is measured over time, cells can be often tracked from Annexin V and 7-AAD negative (viable, or no measurable apoptosis), to Annexin V positive and 7-AAD negative (early apoptosis, membrane integrity is present) and finally to Annexin V and 7-AAD positive (end stage apoptosis and death). The movement of cells through these three stages suggests apoptosis. In contrast, a single observation indicating that cells are both Annexin V and 7-AAD positive, in of itself, reveals less information about the process by which the cells underwent their demise.

Purified Annexin V is routinely tested by flow cytometric analysis. Other applications were tested at BD Biosciences Pharmingen during antibody development only or reported in the literature.