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’ Swine 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 Swine 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.
- Prepare samples and controls.
- Dilute 10X Binding Buffer 1:10 WITH DiH2O.
- Wash cells twice in ice-cold culture medium or PBS and resuspend in ice-cold 1X Binding Buffer at 5 x 105 – 1 x 106 cells/mL.
- Reconstitute Swine Annexin V-Fluorescein in 1 mL Annexin Reconstitution Buffer, 1X.
- Dilute reconstituted Annexin V-Fluorescein 1:5 in PBS.
- Add diluted Annexin V-Fluorescein to each sample at ~1:10.
- Dilute PI 1:10 in PBS.
- Add diluted PI to each sample at ~1:20.
- Incubate 15 minutes on ice and protected from light.
- Add 400 µL 1X Binding Buffer to each sample.
- Analyze with a flow cytometer. Annexin V-Fluorescein excites at 494 nm and emits at 521 nm. PI excites at 536 nm and emits at 617 nm.