Mitochondrial permeability transition (MPT), the collapse of electrochemical gradient across the mitochondrial membrane, is a one of the early events during cellular apoptosis. Mitochondrial dysfunction or injury is the hallmark of many diseases like cerebral ischemia, neuronal diseases, reperfusion injury and cancer. Key factors regulating MPT include calcium, the cellular redox status (including levels of reactive oxygen species) and the mobilization and targeting to mitochondria of Bcl-2 family members. The measurement of mitochondrial membrane potential is one of the methods used to study signaling mechanisms involved in the initiation of the apoptotic cascade.
The JC-1 Mitochondrial Membrane Potential Detection Kit™ from Cell Technology Inc. uses a unique fluorescent cationic dye, JC-1, to measure the collapse of the electrochemical gradient across the mitochondrial membrane. In our studies, we were trying to dissect the molecular mechanisms underlying the apoptotic activity of camptothecin in MCF-7 cells. We treated MCF-7 cells with 10 uM camptothecin for 24 hours and then used the JC-1 kit to assay the effects of the 10 uM camptothecin on mitochondrial membrane potential. The kit is extremely simple and easy to use. It gives highly sensitive and reproducible results across multiple experiments. The biggest advantage of the kit is that it is adaptable to many formats. Whereas most labs have detected the fluorescent signal by flow cytometry, the kit is also compatible with fluorimetric plate readers and fluorescence microscopy. We used fluorescent microscopy to analyze the results of our experiments.
The JC-1 dye accumulates in the mitochondria of healthy cells as aggregates, which are fluorescent red in color. Upon the onset of apoptosis, the mitochondrial potential collapses and the JC-1 dye can no longer accumulate in the mitochondria and remains in the cytoplasm in a monomeric form which fluoresces green. The differential distribution of the red and green forms of the dye is easily analyzed by fluorescence microscopy. The dye is highly specific and sensitive and gives very little background.
The kit has enough reagents for 100 reactions and has a good shelf life of about a year at 4ºC. It is also very reasonably priced. The technical manual is detailed and self-explanatory. No tricks are required to make the protocol work, which is one of the great things about this kit. The kit is compatible with both adherent and suspension cells, and harvesting the cells by scraping does not give any increased background.
I cannot think of a negative aspect of the JC-1 kit. The technical support of the company is excellent. The merit of the kit is further confirmed by the numerous times it has been cited in research papers. I would highly recommend the kit to all researchers working in the field of mitochondrial physiology and apoptosis.