Annexin V-FITC Apoptosis Assay Kit

1. Prepare samples and controls, 2. Dilute 10X Apoptosis Wash Buffer 1:10 with diH2O., 3. Reconstitute FLICA with 50 µL DMSO., 4. Dilute FLICA 1:5 by adding 200 µL PBS., 5. Add diluted FLICA to each sample at 1:30-1:60 (e.g. spike at 1:30 by adding 10 µL to 290 µL cultured cells)., 6. Incubate approximately 1 hour., 7. Wash and spin cells three times., 8. If desired, label with additional stains, such as Hoechst, DAPI, or an antibody., 9. If desired, fix cells., 10. Analyze with a fluorescence microscope or flow cytometer. FLICA 660 excites at 660 nm and emits at 680-690 nm.

Kit 9124:, Annexin V-FITC, 200X, 50 µL, 1 vial, #6340, 10X Binding Buffer, 1.7 mL, 3 vials, #6341, Propidium Iodide, 250 µg/mL, 1 mL, 1 vial, #638, Kit Manual

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., , With our Annexin V-FITC Apoptosis Assay Kit, ImmunoChemistry Technologies provides a proven method for quickly and easily distinguishing two populations of dying cells from viable cells using Annexin V-FITC and Propidium Iodide (PI) reagents. Cells in the early stages of apoptosis with intact cell membranes and surface -exposed PS will stain positive for Annexin V-FITC. PI is included in the apoptosis assay kit to identify late apoptotic and necrotic cells, which have lost plasma membrane integrity. These cells will become dually labeled with Annexin V-FITC and Propidium Iodide (green and red fluorescence). Live cells with intact plasma membranes will exclude PI and will remain unstained by the Annexin V-FITC 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.