LipidSpot™ Lipid Droplet Stains

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.

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.

Most of our MitoView™ dyes are sensitive to mitochondrial membrane potential to some degree, and therefore are recommended for use in live cells only.

Our MitoView™ Fix 640 dye is designed to be fixable after staining of live cells. Staining is compatible with formaldehyde fixation, detergent permeabilization, methanol fixation, and subsequent immunofluorescence staining. MitoView™ Fix is not recommended for staining cells that are already fixed, because staining is non-specific in fixed cells.

MitoView™ Green is relatively insensitive to mitochondrial membrane potential, and can be used to stain formaldehyde-fixed cells (for best results, we recommend first fixing, then staining). MitoView™ Green does not stain well with samples that are fixed with methanol, permeabilized with detergent, or paraffin embedded. Nonyl Acridine Orange also stains mitochondria in fixed cells, but the staining of fixed cells is not specific to mitochondria (cytoplasmic and nuclear staining is also observed). In general, staining of fixed cells with mitochondrial dyes is less specific than live cell staining. For best results when staining mitochondria in fixed cells or tissue sections, we recommend using one of our Mitochondrial Marker Antibodies, available with a wide selection of bright and photostable CF® Dyes and other conjugations.