The MitoProbe™ JC-1 Assay Kit is a quick and reliable assay used to detect changes in mitochondria by flow cytometry.Selective to mitochondrial changesThe membrane-permeant JC-1 dye is widely used in apoptosis studies to monitor mitochondrial health. JC-1 dye exhibits potential-dependent accumulation in mitochondria, indicated by a fluorescence emission shift from green (~529 nm) to red (~590 nm). Consequently, mitochondrial depolarization is indicated by a decrease in the red/green fluorescence intensity ratio. The potential-sensitive color shift is due to concentration-dependent formation of red fluorescent J-aggregates.Unlike other mitochondrial stains that are affected by plasma membrane potential, the ratio of green to red fluorescence of JC-1 depends only on the membrane potential and not other factors such as mitochondrial size, shape, and density, which may influence single-component fluorescence signals. Use of fluorescence ratio detection allows researchers to make comparative measurements of membrane potential and determine the percentage of mitochondria within a population that respond to an applied stimulus.Indicator of early apoptosisA distinctive feature of the early stages of programmed cell death is the disruption of active mitochondria. This mitochondrial disruption includes changes in the membrane potential and alterations to the oxidation-reduction potential of the mitochondria. Changes in the membrane potential are presumed to be due to the opening of the mitochondrial permeability transition pore (MPTP), allowing passage of ions and small molecules. The resulting equilibration of ions leads in turn to the decoupling of the respiratory chain and the release of cytochrome c into the cytosol.Simple, streamlined protocolOne of the biggest challenges to working with dual-emitting dyes like JC-1 is compensation. The MitoProbe™ JC-1 Assay Kit provides a mitochondrial membrane-potential disrupter, CCCP, in order to produce compensation controls leading to a correctly compensated green-to-red fluorescence ratio. The stain protocol is simple and straightforward: incubate the cells with JC-1 dye and then analyze the fluorescent signal in the FITC and PE channels of of a 488 nm laser.