MitoGLO™ JC-1 Dye Optimized for Cell Culture

Summary of the Cell Death Field
The cell death field continues to emerge as it moves beyond classical distinctions between apoptosis (a form of PCD) and necrosis, an accidental cell death. Apoptosis has been characterized by morphological changes including cell shrinkage, chromatin condensation, and cell disintegration into small “apoptotic bodies” which can be removed in vivo by phagocytosis. Apoptotic cascades can be initiated through two major pathways: (1) Activation of death receptor family members in response to ligand binding, or (2) Release of cytochrome c from the mitochondria. Initiation of the apoptotic program through either signal transduction pathway leads to caspase activation and massive protein cleavage, resulting in cell demise and ultimate death. In contrast, necrosis has been characterized by cellular swelling and disruption of the plasma membrane, leading to release of cellular components and in vivo, an inflammatory tissue response. There is now considerable evidence indicating that the classical distinction between apoptosis or PCD and necrosis is a simplification of complex death processes. For example, data suggests that cell death can occur as a program in the absence of caspase activation. Alternative models of PCD that have been described include autophagy, paraptosis, mitotic catastrophe, apoptosis-like PCD, and necrosis-like PCD.

Mitochondrial Membrane Potential and PCD

Mitochondria have long been known to be critical for overall cell survival because of their role in energy metabolism. However, it wasn’t until the mid 1990’s that it became evident that mitochondria are also active participants in apoptosis/PCD. Energy released during mitochondrial respiration is stored as a negative electrochemical gradient across the mitochondrial membrane, and the mitochondrial membrane potential DY is polarized. Collapse or depolarization of the DY is often, but not always, one of the first measurable events during apoptosis/PCD, and may even be a prerequisite for cytochrome C release. However, the relationship between cytochrome C and depolarization of DY has yet to be fully elucidated. In addition to apoptosis, changes in the DY have been reported during necrosis (depolarization)2 and cell cycle arrest (hyperpolarization)5. Emerging knowledge about how the DY changes during apoptosis/PCD, necrosis, and the cell cycle is helping to gain insight into the role of the mitochondria during cell death and other cellular processes.

MitoGLO™, JC-1 Dye Optimized for Cell Culture. Mitochondrial depolarization can be detected by a unique fluorescent cationic dye, 5,5',6,6'-tetrachloro-1,1',3,3'-tetraethyl-benzamidazolocarbocyanin iodide, also known as JC-1 (reviewed in 6-8). The fluoroscence emission of JC-1 varies depending on the relative concentration of the dye, which in turn is determined by DY. JC-1 which can exist as a monomer or as aggregates, referred to as J-aggregates, excites at 488-490 nm. The monomeric form of the dye emits at 527 nm, whereas the J-aggregates emit at 590 nm. JC-1 dye has been used to monitor DY across the spectrum of species, from plant to worm to human. Once inside the cell, the lipophilic MitoGLO™, bearing a delocalized positive charge rapidly enters the negatively charged mitochondria. As the concentration of MitoGLO™ increases within polarized mitochondria, J-aggregates form which fluoresces red (590 nm). In cells containing mitochondria undergoing depolarization, MitoGLO™ J-aggregates do not remain sequestered within the mitochondria and are distributed throughout the cytoplasm in its monomeric form. This event leads to a decrease in spectral shift and loss of red fluorescence. In some model systems, an increase in green fluorescence may also be observed during the cell death process.

MitoGLO™ Staining in Control and Apoptotic Cells. (A) DMSO carrier treated only Jurkat cells (1x106 cells/mL), and (B) staurosporine (3 h, 37°C) treated Jurkat cells (1x106 cells/ mL), stained with MitoGLO™ according the protocol as described in the Flow Cytometry section and analyzed on a BD FACSCaliber™. In the control population [(DMSO carrier treated only Jurkat cells), (A)], MitoGLO™ staining is seen in both the FL-2 and FL-1 channel although R3 represents only a small percentage of the entire cell population (R3 = 8.08%). (B) Staining is seen in both the FL-2 and FL-1 channel, however there is a significant shift to R3 (R3 = 38.13%) indicating lowered red fluorescence which is consistent with deplorization of the DY.

MitoGLO™ Staining in Jurkat cells. Jurkat cells were stained and viewed through a fluorescence microscope using a broad band path filter. Non-apoptotic cells exhibit orange stained mitochondria (2 cells at left). Apoptotic cells at varying stages of mitochondrial DY appear green (3 cells at right).