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What is the staining mechanism of MitoView™ and other mitochondrial dyes? Where do they localize in the organelle?

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Cationic dyes are widely used as mitochondrial probes. They accumulate within the cell and preferentially localize in the mitochondrial matrix, induced by the greater negative membrane potential of mitochondria in live cells compared to the plasma membrane potential. Membrane potential plays a direct role in governing the distribution of the dyes across the plasma membrane: the more negative the potential, the greater the accumulation of the positively charged dyes. MitoView™ 633 is a cationic lipophilic dye that is potential-dependent and accumulates in mitochondria in proportion to the electron gradient similar to classic dyes like TMRM, TMRE and Rhodamine-123.

Ratiometric dyes like JC-1 constitute another class of potential-dependent mitochondrial dyes. In normal healthy cells, JC‐1 accumulates in the mitochondria and undergoes aggregation in a potential‐dependent manner, whereas in unhealthy, apoptotic or dying cells with dissipated mitochondrial potential, the dye delocalizes to the cytosol where it dissociates to the monomeric state. The aggregated dyes (J-aggregates) fluoresce red, while the monomeric dyes fluoresce green.

Some mitochondrial‐specific dyes are considered “structural” dyes, as opposed to “functional” dyes, because they are capable of staining mitochondria regardless of their polarization status. The fluorescence of cells stained with these dyes is directly proportional to their mitochondrial content or “mitochondrial mass.” MitoView™ Green is a potential-independent dye that accumulates in the mitochondria and interacts with the mitochondrial matrix via hydrophobic interactions. It can be used to stain mitochondria in live as well as formaldehyde-fixed cells. MitoView™ Green staining is not compatible with solvent-based fixatives or permeabilization. Nonyl Acridine Orange (NAO) is another potential-independent mitochondrial dye that binds specifically to cardiolipin in the inner mitochondrial membrane. However, mitochondrial dye staining of fixed cells generally is less specific compared to live cell staining. For fixed cell staining, we recommend using one our CF® Dye conjugated mitochondrial marker antibodies.

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