Annexin-5 apoptosis assay
Annexin-5 is a member of a highly conserved protein family that bind acidic phospholipids in a calcium-dependent manner. Annexin-5 is known also as 35 kDa Calelectrin, 35-gamma Calcimedin, Anchorin C2, PAP-1 (placental anticoagulant protein-1), Calphobindin-1, Endonexin-2, Lipocortin V, VAC-alpha (vascular anticoagulant-alpha).
The protein has been shown to possess a high affinity for phosphatidylserine. Phosphatidylserine is translocated from the inner side of the plasma membrane to the outer layer when cells undergo death by apoptosis or cell necrosis and serves as one of several signals by which cell destined for death are recognized by phagocytes (see also: apoptotic bodies) (Van Engeland et al, 1998).
Cell death by apoptosis and necrosis differ from each other in that cell membranes maintain their integrity during the initial stages of apoptosis, while they become leaky during necrotic cell death. If Annexin-5 binds to the cell surface this indicates that cell death is imminent. In order to differentiate apoptosis from necrosis, a dye exclusion test with propidium iodide is performed to establish whether membrane integrity has been conserved or whether membranes have become leaky. A combination test measuring Annexin-5 binding and dye exclusion thus allows discrimination between intact cells, apoptotic cells, and necrotic cells. The test allows detection of apoptosis at early stages before gross morphological changes have occurred and has been adapted for use with flow cytometry, light and electron microscopy and can be applied to vital and fixed material (Miller, 2004; Pellicciari et al, 1997). Schellenberger et al (2002) have conjugated Annexin-5 to crosslinked iron oxide (CLIO) nanoparticles, a functionalized superparamagnetic preparation developed for target-specific magnetic resonance imaging (MRI). The resulting nanoparticles can be used to remove apoptotic cells from cell suspensions containing healthy and apoptotic cells by magnetic column chromatography and to visualize apoptotic cells by MRI.
Dillon et al (2000) have reported that detection of annexin-5 binding may not always be indicative of apotosis. Phosphatidylserine exposed on the majority of B-cells in vivo does not reflect early apoptosis, but, instead, plays a role in receptor-mediated signaling events.
For other assays allowing detection of apoptotic cells see also: apoptosis assays. Kawaminami et al (1998) have reported that the withdrawal of ovarian hormones induces huge castration cells in the anterior pituitary gland. These cells express annexin-5. Annexin-5 is detected also in follicular epithelial cells and parafollicular cells of the thyroid gland, adrenocortical cells of the zona fasciculata and zona reticularis, luteal cells, testicular interstitial cells, and Sertoli cells. It is not detected in the pineal gland, the parathyroid gland, the islet of Langerhans, the adrenal medulla, zona glomerulosa cells, and granulosa cells. The authors have suggested that annexin-5 contributes to secretory cell functions, which may be common to endocrine cells secreting chemically different hormones.