MitoSpy™ OrangeMitoSpy™ 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.

annexin image
annexin v alexa fluor
annexin v bv421

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.