JC-1 is widely used for determining mitochondrial membrane potential with flow cytometry. It is capable of entering selectively into mitochondria, and changes reversibly its color from green to orange as membrane potentials increase (over values of about 80-100 mV). This property is due to the reversible formation of JC-1 aggregates upon membrane polarization that causes shifts in emitted light from 530 nm (i.e., emission of JC-1 monomeric form) to 590 nm (i.e., emission of J-aggregate). When excited at 490 nm, the color of JC-1 changes reversibly from green to greenish orange as the mitochondrial membrane becomes more polarized. Both colors can be detected using the filters commonly mounted in all flow cytometers, so that green emission can be analyzed in fluorescence channel 1 (FL1) and greenish orange emission in channel 2 (FL2). The main advantage of the use of JC-1 is that it can be both qualitative, considering the shift from green to orange fluorescence emission, and quantitative, considering the pure fluorescence intensity, which can be detected in both FL1 and FL2 channels. Besides its wide use with flow cytometry, it is also used in fluorescence imaging. We have developed a protocol to use it in fluorescence microplate platform. Although JC-1 is widely used in many labs, its poor water solubility makes it hard to use for some applications. Our JC-10 has much better water solubility than JC-1, and in some cell lines JC-10 has even superior performance to JC-1. Interestingly the performance of JC-10 is quite cell line-dependent.
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