Detect apoptosis and membrane permability with the Annexin V-FITC Apoptosis Assay Kit. This in vitro apoptosis assay employs the green fluorescent Annexin V-FITC reagent to label exposed phosphatidylserine in cultured cell samples. Detect membrane compromised cells, a trademark of late apoptosis or cell necrosis, with the live/dead stain, propidium iodide. Analyze the fluorescent signals by flow cytometry.
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.
Apoptosis, Exposed phosphatidyl serine
488 nm / 530 nm
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- Prepare samples and controls
- Dilute 10X Binding Buffer 1:10 with diH2O.
- Wash cells in ice-cold culture medium or PBS and resuspend in ice-cold 1X Binding Buffer.
- Dilute Annexin V-FITC 1:10 in PBS.
- Dilute Propidium Iodide (PI) 1:2 in PBS.
- Add diluted Annexin V-FITC to each sample at a ratio of 1:20 (5 µL per 100 µL aliquot of cultured cells).
- Add diluted PI to each sample at a ratio of 1:20 (5 µL per 100 µL aliquot of cultured cells).
- Incubate ~10 minutes, protected from light.
- Dilute samples by adding 250 µL 1X Binding Buffer to each.
- Analyze with a flow cytometer. Annexin V-FITC optimally excites at 494 nm and emits at 519 nm (typically read in FL1). PI optimally excites at 536 nm and has a peak emission at 617 nm (typically read in FL3).