The study of apoptosis, or programmed cell death, is one of the most cutting-edge research areas in modern biology. Apoptosis is a multi-step process characterized by the orchestrated collapse of the cell involving membrane blebbing, cell shrinkage, protein fragmentation, chromatin condensation and cleavage of DNA. The measurement of apoptosis can be done at any one of these steps. The loss of membrane integrity is one of the early steps of the apoptotic cascade and leads to the translocation of a phosphatidylserine from the cytoplasmic side of the cell to the outer membrane. The Annexin-FITC Apoptosis Detection Kit I is designed for the measurement of early apoptosis. The kit uses Annexin V, a calcium dependent phosphatidylserine binding protein, conjugated to the FITC fluorochrome. The Annexin-FITC serves as a sensitive probe for detecting cells in the early stages of apoptosis by flow cytometry.
The biggest advantage of the kit is its sensitivity and simplicity. The protocol is easy to follow and is highly sensitive for the detection of Annexin positive cells. The kit can be used for accurate quantitative determinations of the extent of apoptotic cell death induced by therapeutic agents, radiation etc. The kit also contains propidium iodide, a dye used to distinguish between the viable and the non-viable cells. The double staining procedure allows us to quantify cells in early as well as late stages of apoptosis. It also detects cells dying by necrosis and thereby helps to eliminate any false positives in the cell samples. Some versions also use 7-AAD instead of PI. The kit can be used for a large number of samples and has a good shelf life.
The Annexin-FITC kit is best suited for studying apoptosis in suspension cells. The cells have to be cautiously handled; any harsh treatment during the annexin-FITC staining procedure may disrupt the membrane integrity of the cells. The disruption of the cell membranes may expose the phosphatdylserine residues, which on binding to annexin-FITC will result in non-specific background. One has to be especially careful in staining serum-starved suspension cells which are very fragile and tend to give high background staining with annexin-FITC. The biggest disadvantage of the kit is that it cannot be used very well with adherent cells, since trypsinization or scraping (used to harvest the cells) may disrupt the membrane integrity, giving rise to high false positives even in the control populations. Therefore, experiments must be designed while keeping the limitations of the kit in mind.
Overall, the annexin-FITC apoptosis kit is a highly convenient method of quantitating early apoptosis in cells. The kit is economical, easy to use and comes with enough reagent to analyze many samples. The kit has some limitations especially when used for studies involving adherent cell lines. However, several protocols and reagents are available which can circumvent some these difficulties. I would highly recommend this kit as a valuable tool for the measurement of apoptosis in normal and diseased cell states.
Piyali Dasgupta
Postdoctoral Fellow
H. Lee Moffitt Cancer Research Center
Tampa, Florida