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INTRODUCTION
Apoptosis is a well-documented phenomenon in many cellular systems.1 It plays a key role in tissue and organ development during embryogenesis as well as in regulating cell turnover in adult tissues. In the immune system, apoptosis provides the mechanism for deletion of autoreactive T cells in the thymus 2, low responsive B lymphocytes in germinal centers 3, and of target cells susceptible to attack by cytotoxic T lymphocytes and natural killer cells.1, 4 Structurally, apoptosis is characterized by chromatin condensation, reduction in cell size, and endonuclease cleavage of DNA into nucleosomal size fragments.1,4,5 In addition to these changes in cell morphology that occur during
apoptosis, a major event is the loss of membrane phospholipid asymmetry, with translocation of phosphatidylserine (PS) from the inner leaflet of the phospholipid bilayer to the cell surface.6 While the function of PS externalization is unclear, it may serve as a “signal" for recognition by phagocytic cells which respond by engulfing the apoptotic cell
before loss of plasma membrane integrity. Exposure of PS on the cell surface provides a simple means for detecting cells undergoing apoptosis. Annexin V (Mr 36-kDa), a member of the annexin family of calcium-dependent phospholipid binding proteins, has a high affinity for PS-containing phospholipid bilayers.7,8 When PS is exposed on the extracellular face of a cell membrane, annexin V binds with an affinity of approximay 7 nM.9 Fluorochrome conjugates of annexin V can be used to monitor changes in cell membrane phospholipid asymmetry 10-13, thereby providing a convenient tool for
detection of apoptotic cells (Figure 1). Since externalization of PS occurs earlier than the nuclear changes associated with apoptosis, the ApoScreen Annexin V Apoptosis Kit can be used for detection of cells earlier in the apoptotic pathway than do DNA-based assays.14 Labeled annexin V can also be used to analyze changes in membrane
asymmetry in plaets 10 and erythrocytes.13 For individual ApoScreen labeled annexin V reagents see product nos. 10040-02 and 10040-09.
PRINCIPLE OF THE ASSAY
The ApoScreen Annexin V Apoptosis Kit employs a R-phycoerythrin-labeled annexin V (annexin V-R-PE) in concert with
7-AAD to evaluate subpopulations of cells undergoing apoptosis. During the early stages of apoptosis, cells begin to display PS on the outer cell membrane where it is readily detectable by staining the cells with annexin V-R-PE. As the
plasma membrane becomes increasingly permeable during the later stages of apoptosis, 7-AAD can readily move across the cell membrane and bind to cellular DNA, providing a means for identifying those cells that have lost membrane integrity through mechanisms including necrosis. When cells are double stained with annexin V-R-PE and 7-AAD, three different cell populations may be observed (Figure 2): (i) live cells that do not stain with either annexin VR-PE or 7-AAD; (ii) necrotic cells that stain with both reagents; and (iii) apoptotic cells that stain with annexin V-R-PE only. Analysis can be performed on any flow cytometer equipped with a single laser using excitation at 488 nm.
KIT COMPONENTS
? 1 mL annexin V-R-PE: Store at 2-8°C. Use as directed; no pre-dilution necessary.
? 1 mL 7-AAD (100 tests): Store at 2-8°C. Use as directed; no pre-dilution necessary. Avoid skin contact and dispose of properly.
? 7 x 1.5 mL 10X binding buffer: Store at 2-8°C. Dilute 1 part 10X buffer with 9 parts dH20 prior to use.