Annexin V anti-Human, V500, Clone: V500, BD

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 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™ V500
• 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, V500 Annexin V staining can identify apoptosis at an earlier stage than assays based on nuclear changes such as DNA fragmentation
• V500 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 V500 Annexin V is typically used in conjunction with a vital dye such as propidium iodide (PI) or 7-Amino-Actinomycin (7-AAD) to allow the investigator to identify early apoptotic cells (PI negative, V500 Annexin V positive)
• Viable cells with intact membranes exclude PI, whereas the membranes of dead and damaged cells are permeable to PI
• For example, cells that are considered viable are both V500 Annexin V and PI negative while cells that are in early apoptosis are V500 Annexin V positive and PI negative, while cells that are in late apoptosis or already dead are both V500 Annexin V and PI 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 V500 Annexin V and PI
• However, when apoptosis is measured over time, cells can be often tracked from V500 Annexin V and PI negative (viable, or no measurable apoptosis), to V500 Annexin V positive and PI negative (early apoptosis, membrane integrity is present) and finally to V500 Annexin V and PI 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 V500 Annexin V and PI positive, in of itself, reveals less information about the process by which the cells underwent their demise
• When compensating dyes in this spectral range (such as Horizon™ V500 and AmCyan), the most accurate compensation can be obtained using single stained cellular controls
• Due to spectral differences between cells and beads in this channel, using BD CompBeads can result in spillover errors for V500 and AmCyan reagents
• Therefore, the use of BD CompBeads or BD CompBeads Plus to determine spillover values for these reagents is not recommended
• Different V500 reagents (e.g
• CD4 vs
• CD45) can have slightly different fluorescence spillover therefore, it may also be necessary to use clone specific compensation controls when using these reagents.