The Annexin V-FITC Apoptosis Detection Kit by Oncogene Research Products utilizes a well-established system to differentiate between necrotic and apoptotic cells. In this system, the anti-coagulant annexin V is used (in the presence of calcium) to bind to phosphatidyl serine (PS). PS is normally a cytoplasmic protein, but it is translocated to the cell surface in the early stages of apoptosis. Because the annexin V is conjugated to fluorescein isothiocyanate (FITC), it can be detected using flow cytometry or fluorescence microscopy. Necrotic cells exhibit membrane permeabilization and will also bind Annexin V-FITC, but propidium iodide is used to distinguish between viable, early apoptotic and necrotic or late apoptotic cells. Propidium iodide is excluded from viable (FITC-negative) and early apoptotic (FITC-positive) cells. Late apoptotic cells will stain with both FITC and propidium iodide due to the final necrotic-like disintegration of the cell.
I have extensive experience with the Annexin-FITC Apoptosis Detection Kit and have used it on several different cell lines derived from rat, mouse and human. Apoptosis was induced using individual cytokines, cytokines mixtures or exogenous NO-donors. I was pleased with the Annexin-FITC kit and found that it gave accurate results when compared to other apoptosis-detection methods, such as staining for DNA content in late apoptosis with propidium iodide. And it is easy to use. Cells can be stained in culture media following the RAPID protocol or washed in PBS and assayed using the conventional method. I found that anything that was not provided in the kit itself (PBS, pipetters, etc.) was easily found in the lab.
However, there are some things to be aware of when using this kit. For instance, it is important to be aware of time restrictions when performing this assay. The assay must be performed quickly, as apoptosis is an ongoing process and does not stop when the cells are being processed. Samples need to be read immediately after processing as FITC signal loss can occur within 1 hour. One also needs to be careful with the handling of samples as the manipulation of the cells with pipetting can significantly alter results (ie. Increase the number of necrotic cells). Also, this assay cannot distinguish between late apoptotic and necrotic cells.
In summary, I have found the Annexin-FITC Apoptosis Detection Kit to be accurate and easy to use. It is important to be aware, however, that the manipulation of adherent cells can result in variable results. Also, although this kit can detect cells that are in the early and late stages of apoptosis, it does not differentiate between late apoptosis and necrosis.
Karrie Comatas
Research Technician
Duke University