This kit is intended for early detection of apoptotic cells by flow cytometry or immunoflourescent cytology. In normal cells phosphatidylserine (PS) is located on the inner leaflet of the plasma membrane. During the early stages of apoptosis, PS is translocated to the outer layer and is exposed on the external surface of the cell. This early event in apoptosis can be detected by using a sensitive method in which to detect PS exposure. Translocation of PS to the external surface of the plasma membrane is not a unique property of apoptotic cells, as this phenomenon also occurs during cell necrosis. However, during apoptosis, the cell membrane remains intact; whereas, during necrosis, the cell becomes leaky and loses its integrity. Therefore, it is necessary to assess membrane integrity together with PS translocation.
Annexin V is a Ca 2+ -dependent phospholipid binding protein. This protein can bind to a variety of phospholipids, but it has the highest affinity for phosphatidylserine (PS). Based on its affinity for PS, Annexin V can be utilized as a sensitive probe for cell surface exposure of PS. To use the Annexin V protein as a probe for apoptotic cells, the protein has been labeled with fluorescein isothiocyanate (FITC). In this form, the protein can be used directly for quantification of apoptotic cells. The measurement of Annexin V binding when performed simultaneously with a dye exclusion test (such as propidium iodide) can be used to effectively discriminate between apoptotic and necrotic cells.
Annexin V binds to negatively charged phospholipid surfaces in a Ca(2+)-dependent manner. it prevents the formation of the prothrombinase complex, thereby inhibiting formation of thrombin (anticoagulant activity). As a result, Annexin V causes a dose-dependent prolongation of the clotting time when measured in the modified prothrombin time test. Annexin V also exhibits anti-phospholipase activity.
Applications:Suitable for use in detecting apoptotic cells by flow cytometry or immunofluorescent cytology. Other applications not tested.
Kit Components:*A2296-20D1: Annexin V (FITC) 1x500ul (Sufficient for 50 tests.) Source: E. coli Molecular Weight: 35.8kD Purity: >98% pure as demonstrated by SDS-gel electrophoresis and reverse phase HPLC. Supplied as a liquid in 50mM Tris, 100mM sodium chloride, pH 7.4, 1% BSA, 0.01% sodium azide
A2296-20D2: Binding Buffer, 2X, 1x16ml Dilute in deionized water. Supplied as a liquid from 20mM Hepes/NaOH, pH 7.4, 280mM sodium chloride, 5mM CaCl2
A2296-20D3: Propidium Iodide 1x500ul Supplied as a liquid
Concentration: 20ug/ml
Storage and Stability:Aliquot and store *A2296-20D1 at -20°C. Store other components at 4°C. Stable for 6 months after receipt. For maximum recovery of product, centrifuge the original vial after thawing and prior to removing the cap.
