Apoptosis, or programmed cell death, is a normal physiological event necessary for eliminating unnecessary, unwanted cells from the body. Over the past few years, it has become one of the most researched topics in biology. One of the hallmark events that occurs early in apoptosis is the flipping of phosphatidyl serine from the inner leaflet to the outer leaflet of the cytoplasmic membrane. Annexin V is known to bind tightly to phosphatidyl serine, which makes it ideal for detecting membrane changes associated with apoptosis, using either flow cytometry or in situ detection.
Trevigen's TACS Annexin V Biotin Kit allows for this cell membrane detection while providing flexibility in labeling by using strept-avidin conjugated to any fluorophore of interest. The kit can be used for flow cytometry or in situ detection of suspended cells, adherent cells, or tissues. The protocols provided with the kit are fairly straight forward, easy to follow, and very quick to perform.
We use the TACS Annexin V Biotin Kit in our lab for the detection of apoptosis, or lack thereof, in the cerebellum and hippocampus of 8 – 30 day postnatal Sprague-Dawley rats. We generally perform our Annexin V Biotin protocol on freshly obtained tissue samples. We are also beginning to use Annexin V Biotin in glial cell cultures. After the freshly obtained tissue samples have incubated in the Annexin V Biotin, and have been fixed, then the tissue can be labeled with any strept-avidin fluorophore (which is not provided with the kit), or it can be used for immunohistochemistry/immunocytochemistry. We generally conduct double labeled immunohistochemistry experiments using the TACS Annexin V Biotin Kit along with antibodies such as caspase 3 or CAD.
When analyzing results from the Annexin V Biotin labeling kit, it is important to keep in mind that it can detect cells that are in both late apoptosis as well as early apoptosis. This problem can be overcome by utilizing products such as propridium iodide, which is included with the kit, but we do not use it for our specific research in our lab. This problem can be beneficial in that it has the ability to detect apoptosis that is undetected by TUNEL because DNA fragmentation has not occurred yet. Also, tissue edges may become damaged due to tissue retrieval procedures and not because of apoptosis, which can cause false positive labeling. Again, this should also be taken into account when analyzing results.
Another problem with the kit, as mentioned above, is that it does not come with any strept-avidin fluorophores, so an extra expense must be incurred. Also, the protocols do not provide for any positive controls to ensure the procedure is working properly. So one must use an agent, such as staurosporine, that is known to cause apoptosis before applying the Annexin V Biotin in order to have a positive apoptotic slice to compare to your system of interest.
Our lab has used the Trevigen TACS Annexin V Biotin Kit for over a year now. Although the kit does have its limitations, we have been able to obtain reliable, reproducible results with it. It is well suited for in situ apoptosis detection while many kits require, or are best suited for, the use of flow cytometry. Although the kit is fairly expensive, it is easy to use, and comes with enough of the reagents to run as many as 250 tests (if it is used for flow cytometry rather than in situ as we use in our lab). I would recommend this kit be used as a valuable tool in any apoptotic research study, but I would caution that it not be used as the sole source of apoptosis detection and caution in analysis procedures.
VelvetLee Finckbone
Lab Technician
Dr. Jean Strahlendorf's Lab
Dept. of Physiology
Texas Tech University Health Science Center