Regulatory Status Legend

Description

Apoptosis is a normal physiologic process that occurs during embryonic development as well as in maintenance 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 asymmetry 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 including BD Horizon™ BV711. This format retains its high affinity for PS and thus serves 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, BV711Annexin V staining can identify cells undergoing apoptosis at an earlier stage rather than assays based on nuclear changes such as DNA fragmentation.

BV711 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 BV711 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 (BV711 Annexin V positive, 7-AAD negative). Viable cells with intact membranes exclude 7-AAD, whereas the membranes of dead and damaged cells are permeable to the nucleic acid dye, 7-AAD. For example, cells that are considered viable are both BV711 Annexin V negative and 7-AAD negative while cells that are in early apoptosis are BV711 Annexin V positive and 7-AAD negative. Cells that are in late apoptosis or already dead are both BV711 Annexin V positive 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 BV711 Annexin V and 7-AAD. However, when apoptosis is measured over time, cells can be often tracked from being BV711 Annexin V negative and 7-AAD negative (viable, or no measurable apoptosis), to BV711 Annexin V positive and 7-AAD negative (early apoptosis, membrane integrity is present), and finally to BV711 Annexin V positive 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 BV711 Annexin V and 7-AAD positive, by itself reveals less information about the process by which the cells underwent their demise.

The Annexin V is conjugated to BD Horizon BV711 which is part of the BD Horizon Brilliant™ Violet family of dyes. This dye is a tandem fluorochrome of BD Horizon BV421 with an Ex Max of 405-nm and an acceptor dye with an Em Max at 711-nm. BD Horizon BV711 can be excited by the violet laser and detected in a filter used to detect Cy™5.5 / Alexa Fluor® 700-like dyes (eg, 712/20-nm filter). Due to the excitation and emission characteristics of the acceptor dye, there may be moderate spillover into the Alexa Fluor® 700 and PerCP-Cy5.5 detectors. However, the spillover can be corrected through compensation as with any other dye combination.

When compensating dyes in this spectral range, the most accurate compensation can be obtained using unstained and single color-stained cellular controls.