APOPercentage™ Apoptosis Assay From Biocolor

The APOPercentage™ Apoptosis Assay is a detection and measurement system to monitor the occurrence of apoptosis in mammalian adherent cells during in vitro culture. It makes use of the ability of the cell membrane to reorganize during apoptosis. The assay uses a dye that is selectively imported by cells undergoing apoptosis. The cells are incubated with the dye for 30 minutes and after washing, they can be analyzed by microscopy which allows photographic evidence, or the uptaken dye can be released from the cells by incubation with the APOPercentage™ Dye Release Reagent followed by the measurement of changes in absorbance in the culture media between apoptotic cells and non-apoptotic cells.

In our lab, we have used this system to detect apoptosis on cell lines and primary cells. Following the method supplied by the manufacturer, cells were grown to confluency and then treated with an apoptotic agent (in our case, we use staurosporine 10 µM for 1 hour); 30 minutes prior to the end of incubation, the APOPercentage™ Dye is added and consequently, the cells are repeatedly washed in PBS until the resulting PBS wash is clear. At that point, the cells can be analyzed by microscopy, producing very clear results. If photography is not possible, the cells can then be treated with the APOPercentage™ Dye Release Reagent for 10 minutes, and the solution collected for an absorbance reading at 550 nm in a spectrophotometer. In both cases, the result was the same. In fact, I took photographs first and then released the dye for spectrophotometric reading.

I found this system very reproducible. It is easy to set up and the manual insert is clear and easy to follow. Although the manufacturer recommends seeding cells onto 96-well plates, I used 24-well plates as I find that format more suitable for our primary cells. The recommended dilution of the dye (1:20) gave nice results, although similar results were obtained with a dilution 1:50. Something to take into consideration is the need for washing the cells really thoroughly after incubating with the dye, as any remaining free dye could interfere with the quality of the experiment.

In short, this is a very easy, straightforward assay which gives the expected outcome in a quick format. The good thing is that it makes possible the detection of a single apoptotic cell in a monolayer, making it a very sensitive system when analyzed by photography. Of course, correct controls are required as with any other system. The possibility of uptaken dye release is good, but I can’t emphasize enough the importance of thorough washing of the cells before applying the release reagent.

I have used this system many times over the last couple of years, and I can say that it works very well and it will give the kind of photographic evidence that many journals will like in their publications.