Mitochondrial dyes, including MitoView™ Mitochondrial Dyes, are positively charged and lipophilic. They passively diffuse across cellular membranes and are presumed to accumulate in the mitochondrial matrix due to the proton gradient in the mitochondria (for a detailed review, see Cytometry Part A79A: 405-425, 2011).
However, some dyes are still retained in mitochondria after depolarization. Our dye chemists hypothesize that this is because some of the dyes are more lipophilic than others. Once they accumulate in the mitochondria because of their charge, they are less likely to diffuse back into the cytoplasm due to their hydrophobicity, even after the proton gradient that attracted them is dissipated by mitochondrial depolarization. Probably they associate with the mitochondrial membranes instead.
The so-called potential-independent dyes like MitoView™ Green, MitoTracker® Green, and Nonyl Acridine Orange are much more hydrophobic than potential-responsive dyes like MitoView™ 633, Rhodamine 123, and JC-1. The former dyes are retained after mitochondrial depolarization, and can be used to measure mitochondrial mass independent of potential. However, it would be more accurate to call these dyes relatively potential-insensitive, rather than potential-independent, because mitochondrial potential still plays a role in their localization. These dyes have been reported to show some loss of signal upon depolarization (Cytometry 39(3):203-10, 2000).
There is another class of mitochondrial dyes that accumulate in mitochondria based on charge, but also have a reactive group that can covalently link the dye to protein targets within the mitochondria, allowing them to be well-retained after fixation and permeabilization. Our MitoView™ Fix 640 is this type of dye.
Some dyes, like MitoView™ Green can stain mitochondria in cells that are already fixed. The mechanism by which this occurs is not well-understood. After fixation, there should be no proton gradient in the mitochondria to attract the dyes at all. Our chemists suspect that there may be some residual membrane potential in fixed mitochondria that is not due to the proton gradient (which would disappear following fixation), but instead arises from uneven distribution of proteins that have different isoelectric points (net charge). There are reports that the net charge of resident proteins in organelles differs based on the pH of the cellular compartment (Proc Natl Acad Sci USA 115(46):11778-11783, 2018). Charge differences may be sufficient to attract cationic lipophilic dyes to mitochondria in the absence of a proton gradient, due to a combination of weak electrostatic and hydrophobic interactions with mitochondrial proteins and membranes.
However, currently there is no direct evidence to suggest this is the mechanism for MitoView™ Green staining of fixed cells. There may be other targets that the dye is binding. For example, Nonyl Acridine Orange is reported to bind cardiolipin, a lipid that is enriched in mitochondrial membranes. It’s possible that MitoView™ Green binds to particular molecules in mitochondria with some degree of specificity. However, staining of fixed cells with mitochondrial dyes generally is not as specific as staining of live cells. That’s why we recommend using mitochondrial marker antibodies instead of dyes to stain fixed cells when possible.View more FAQs