Apoptosis, or programmed cell death, plays an important role in many developmental processes. The detection of apoptotic cells, and monitoring the progression of cells towards apoptosis, are an essential part of our laboratory’s research. Currently, we are studying signaling pathways in mammalian cells that are involved with inducing apoptosis. In our studies, we must be able to verify that the cells are indeed undergoing apoptosis and the Clontech, a Takara Bio Company's ApoAlert Mitochondrial Membrane Sensor Kit is one of the kits we use to detect apoptotic cells during the early stages of apoptosis. This kit is sufficient for approximately 100 assays and the procedure can be done in less than 30 minutes.
The ApoAlert Kit allows us to quickly detect apoptotic cells by detecting the potential changes in the mitochondrial membrane, an early event in apoptosis. The ApoAlert Kit takes advantage of the fact that these apoptosis-induced changes in the mitochondrial membrane result in changes in permeability of the membrane. The Clontech MitoSensor Reagent is a cationic dye sensitive to these changes and, in apoptotic cells, it cannot get into the mitochondria and thus remains in the cytoplasm as monomers. In non-apoptotic cells, it can cross the mitochondrial membrane and thus it aggregates in the mitochondria. The end result is that special dye fluoresces differently in apoptotic cells compared to non-apoptotic cells. In non-apoptotic cells, in which the dye accumulates as aggregates in the mitochondria, the cells show a red fluorescence via fluorescence microscopy. On the other hand, in apoptotic cells, in which the dye stays in the cytoplasm, the cells show a green fluorescence.
The kit can be used for staining both adherent cells and cells in suspension, although we have only used it for staining the former. For staining adherent cells, the cells were first induced to undergo apoptosis after which they were rinsed with cell medium containing no serum. The Clontech MitoSensor Reagent is then diluted in Incubation Buffer (to a final concentration of 5 micrograms/microliter) and centrifuged to remove aggregates. After the spin, the MitoSensor Reagent is added to the cells and incubated. The incubation times will differ for different cell lines, but in our case the incubation went for 20 minutes. The cells are then gently rinsed with culture media without serum. Next, the cells are examined under a fluorescent microscope using a band-pass filter, which detects rhodamine and fluorescein.
As mentioned before, non-apoptotic cells fuoresce an intense red color due to the dye aggregating in the mitochondria. In apoptotic cells, the dye remains as monomers in the cytoplasm and fluoresces a bright green color due to altered membrane potential of the mitochondria.
Overall, the Clontech ApoAlert Mitochondrial Membrane Sensor Kit, one of many kits out there for detecting apoptotic cells, has worked nicely for us. It is very simple, quick to use and, in our hands, cells that have undergone apoptosis are clearly detected with this kit. In addition, one kit is sufficient for performing 100 assays and it is reasonably priced. We have no problems recommending this kit to anyone.
Hee Chul Lee
Department of Biochemistry
NYU School of Medicine