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™ BUV395. 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, BUV395 Annexin V staining can identify cells undergoing apoptosis at an earlier stage rather than assays based on nuclear changes such as DNA fragmentation.

BUV395 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 BUV395 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 (BUV395 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 BUV395 Annexin V negative and 7-AAD negative while cells that are in early apoptosis are BUV395 Annexin V positive and 7-AAD negative. Cells that are in late apoptosis or already dead are both BUV395 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 BUV395 Annexin V and 7-AAD. However, when apoptosis is measured over time, cells can be often tracked from being BUV395 Annexin V negative and 7-AAD negative (viable, or no measurable apoptosis), to BUV395 Annexin V positive and 7-AAD negative (early apoptosis, membrane integrity is present), and finally to BUV395 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 BUV395 Annexin V and 7-AAD positive, by itself reveals less information about the process by which the cells underwent their demise.

The Annexin V was conjugated to BD Horizon BUV395 which is part of the BD Horizon Brilliant™ Ultraviolet family of dyes. This dye is optimal for multicolor flow cytometry because it has little to no spillover into other detectors. With an Ex Max at 348 nm and an Em Max at 395 nm, BD Horizon BUV395 can be excited with a 355 nm laser and detected with a 379/28 filter.

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