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™ PE-CF594. 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, PE-CF594 Annexin V staining can identify cells undergoing apoptosis at an earlier stage rather than assays based on nuclear changes such as DNA fragmentation.

PE-CF594 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 PE-CF594 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 (PE-CF594 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 PE-CF594 Annexin V negative and 7-AAD negative while cells that are in early apoptosis are PE-CF594 Annexin V positive and 7-AAD negative. Cells that are in late apoptosis or already dead are both PE-CF594 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 PE-CF594 Annexin V and 7-AAD. However, when apoptosis is measured over time, cells can be often tracked from being PE-CF594 Annexin V negative and 7-AAD negative (viable, or no measurable apoptosis), to PE-CF594 Annexin V positive and 7-AAD negative (early apoptosis, membrane integrity is present), and finally to PE-CF594 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 PE-CF594 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 PE-CF594 that has been developed for use in multicolor flow cytometry experiments and is available exclusively from BD Biosciences. Annexin is conjugated to BD Horizon PE-CF594, which has been developed exclusively by BD Biosciences as a better alternative to PE-Texas Red®. PE-CF594 excites and emits at similar wavelengths to PE-Texas Red® yet exhibits improved brightness and spectral characteristics. Due to PE having maximal absorption peaks at 496 nm and 564 nm, PE-CF594 can be excited by the blue (488-nm), green (532-nm) and yellow-green (561-nm) lasers and can be detected with the same filter set as PE-Texas Red® (eg, 610/20-nm filter).

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