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-dependant 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-dependant 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, but fixed cell staining tends to be less specific compared to live cell staining. MitoView™ Green staining is also not compatible with solvent-based fixatives or permeabilization. For fixed cell staining, we recommend using one our CF® Dye conjugated mitochondrial marker antibodies. Nonyl Acridine Orange (NAO) is another potential-independent mitochondrial dye that binds specifically to cardiolipin in the inner mitochondrial membrane.

Mounting medium can alter the staining of lipophilic dyes like LipidSpot™, due to interaction of the dyes with glycerol or other components that help form the interface between the coverslip and slide. The antifade compounds in mounting medium are generally compatible with the dyes. In our tests, LipidSpot™ staining was well preserved in EverBrite™ Mounting Medium (catalog. nos. 23001/23002) for up to 24 hours after mounting, but lipid droplet size and staining intensity were somewhat altered after samples were stored in mounting medium for several days. Therefore, if mounting medium is required to image samples, we’d recommend imaging as soon as possible after mounting.

LipidSpot™ is not compatible with FluoroShield mounting medium (staining is lost immediately after mounting). We have not tested other types of mounting medium.

Most of our MitoView™ dyes are sensitive to mitochondrial membrane potential, and therefore can be used in live cells only.

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. For best results staining mitochondria in fixed cells or tissue sections, we recommend using one our Mitochondrial Marker Antibodies, available with a wide selection of bright and photostable CF® dyes and other conjugations.