MitoSpy™ Mitochondrial Probes

MitoSpy™ Orange, MitoSpy™ Red, and MitoSpy™ NIR localize to the mitochondria based on its membrane potential. This potential is created by the transport of ions across the membrane. Thus, MitoSpy™ Orange, MitoSpy™ Red, and MitoSpy™ NIR work well to indicate cell health as well as localization. On the other hand, MitoSpy™ Green FM’s attraction to the mitochondria is not based on membrane potential. While the full mechanism of MitoSpy™ Green FM is not yet understood, it may have to do with homology between the reagent’s structure and proteins at the mitochondrial surface. Because of this reagent’s membrane potential independence, it can be used to measure mitochondrial mass of individual cells in flow cytometry.

MitoSpy™ Orange and MitoSpy™ Red contain a chloromethyl group that covalently attaches to cysteines in the cell. This gives them a higher probability of remaining in a cell after the sample is fixed and permeabilized. However, as the fix/perm process washes out a large amount of the reagent, a much higher concentration of MitoSpy™ Orange and MitoSpy™ Red is required to get sufficient staining.

After the fix/perm process, most MitoSpy™ Green and MitoSpy™ NIR will be washed away. As such, they are not recommended to be used with samples that require fixation and permeabilization.

*Note that all MitoSpy™ products must be used on live samples. The recommendations below reflect whether MitoSpy™ is successfully retained following the "Retention Condition" treatment.

HeLa cells that were stained live with either MitoSpy™ Orange (yellow) or Green (green). Cells that were fixed and permeabilized with 4% PFA and 0.1% Triton X-100 were also stained with DAPI (blue). Photos were taken with a 60x magnification.

MitoSpy™ Orange, MitoSpy™ Red, and MitoSpy™ NIR can also be used as an indicator of cellular health. When the mitochondria are actively respiring, there is a potential difference across the mitochondrial membrane that is called membrane polarization. If a cell is unhappy due to apoptosis or cell death, this probe will not be strongly attracted to the mitochondria of that cell. In the image below, cells positive for MitoSpy™ Orange, MitoSpy™ Red, MitoSpy™ NIR are alive and healthy, while Annexin V positive events are at the beginning stages of apoptosis. In this flow cytometry application, the MitoSpy™ Orange, MitoSpy™ Red, and MitoSpy™ NIR should not be fixed prior to analysis since there is a substantial loss of reagent with fixation.  An apoptotic phenotype is indicated by a low or negative MitoSpy™ Orange, MitoSpy™ Red, or MitoSpy™ NIR signal.  If reagent is lost with fixation, the resulting loss of signal intensity will confound the ability to accurately phenotype apoptosis.  Fixation of MitoSpy™ Orange and MitoSpy™ Red is only applicable for subcellular localization in imaging applications. Fixation is not recommended at all with MitoSpy™ NIR.

Human T-cell leukemia cell line, Jurkat, was treated for 5 hours with LEAF™ purified anti-CD95 (clone EOS9.1), then stained with an impermeant nucleic acid stain, APC, Alexa Fluor® 488, or Brilliant Violet 421™ Annexin V, and either MitoSpy™ Orange, Red, or NIR as indicated. Nucleic acid stain positive events were excluded from analysis.