Swine Annexin V-Fluorescein Apoptosis Assay Kit

1. Prepare the standard and samples in the Sample and Standard Diluent., 2. Add 50 µL of prepared standards and samples in triplicate to appropriate Wells., 3. Add 50 µL of the diluted antibody preparation to the appropriate Wells., 4. Cover plate with Plate Cover and incubate at room temperature (20-25°C) for 1 hour., 5. Dilute 10X Wash Buffer 1:10 with diH2O., 6. Wash plate 4 times with 1X Wash Buffer., 7. Add 100 µL TMB Substrate to each Well., 8. Cover plate with Plate Cover and incubate at room temperature in the dark for 30 minutes to develop the signal., 9. Add 100 µL Stop Solution to each Well., 10. Measure absorbance using a plate reader at 450 nm., 11. Plot the standard curve and calculate sample concentrations.

Kit 9142:, Swine Annexin V-Fluorescein, lyophilized, 1 vial, #6645, Annexin Reconstitution Buffer, 1X, 1 mL, 1 vial, #6639, Annexin Binding Buffer, 10X, 10 mL, 3 bottles, #6640, Propidium Iodide, 250 µg/mL, 1 mL, 1 vial, #638

Annexins are a group of cellular proteins that are mostly found in eukaryotic organisms. To date, more than 150 annexin proteins have been identified in 65 different species. To be classified as an annexin, a protein must meet the following criteria: 1) it has to be capable of binding negatively charged phospholipids in a calcium dependent manner and 2) it must contain a 70 amino acid repeat sequence called an annexin repeat. Annexins are important in various cellular and physiological processes, such as providing a membrane scaffold which is relevant to changes in the physical shape of the cell. Annexin proteins have been shown to be involved in trafficking and organization of vesicles, exocytosis, endocytosis, and calcium ion channel formation. Annexin proteins are not exclusively intracellular proteins; they are also found in the extracellular space and have been linked to several processes, including fibrinolysis, coagulation, inflammation, and apoptosis. Annexin V is a member of a calcium and phospholipid binding family of proteins with vascular anticoagulant activity. Results from in vitro experiments indicate that it may play a role in the inhibition of blood coagulation by competing for phosphatidylserine (PS) binding sites with prothrombin. In healthy cells, PS is usually kept in the inner-leaflet (the cytosolic side) of the cell membrane. When a cell undergoes apoptosis, one of the earliest detectable indicators is the loss of membrane asymmetry. No longer restricted to the cytosolic part of the membrane, PS is translocated to the outer-leaflet and becomes exposed on the surface of the cell. ImmunoChemistry Technologies’ Bovine Annexin V-Fluorescein Apoptosis Assay Kit provides a proven method for quickly and easily distinguishing two populations of dying cells from viable cells using recombinant fluorescein-conjugated Bovine Annexin V and Propidium Iodide (PI). Cells with intact cell membranes and surface-exposed PS, a prominent feature of apoptosis, will stain positive for Annexin V-Fluorescein. PI is included in the kit to identify dying, later stage apoptotic cells which have lost plasma membrane integrity. These cells will become dually labeled with Annexin V-Fluorescein and Propidium Iodide (green and red fluorescence). Live non-apoptotic cells with intact plasma membranes will exclude PI and will remain unstained by the Annexin V-Fluorescein probe, assuming no treatment or cell cycle-associated event temporarily exposes the normally internalized, negatively charged PS entity. The kit also includes a specially formulated, calcium-based binding buffer which is required for Annexin V binding to occur.