Introduction to the Mitochondrial Stress (MitoPT) Kits

Loss of mitochondrial membrane potential could be an indicator of apoptosis. Any cell membrane permeant dye with the capability of being retained within an active (polarized) mitochondrial membrane, and subsequently released upon loss of mitochondrial membrane gradient potential, could serve as a marker of mitochondrial stress. Early on, JC-1 was accepted as a reliable probe for routine screening of different cell populations for mitochondrial polarization status. This potentiometric fluorescent dye enabled scientists to obtain a macroscopic picture of the general health status of different cell populations exposed to varying environmental, immunological, or pharmaceutical treatment regimens. JC-1 (5,5′,6,6′-tetrachloro-1,1′,3,3′ tetraethylbenzimidazolcarbocyanine iodide), dye exhibits the physical characteristics of being a fluorescent lipophilic cation with the ability to form J-Aggregate structures when concentrated within polarized mitochondrial membrane structure. Its ability to perform as a potentiometric fluorescent probe for mitochondrial polarization status assessment can be directly attributed to these physical properties. Its tendency to form J-Aggregate structure when concentrated within polarized mitochondria bestows upon it, the ability to fluoresce at two different emission wavelengths, 527 nm and 590 nm, depending upon its localized concentration. At low cell staining concentrations, JC-1 would exist as the monomeric (green fluorescing) form. When concentrated within inner membrane regions of healthy = polarized mitochondria, JC-1 would exist as the aggregated (J-Aggregate) orange fluorescing form (1).

Mechanism Behind JC-1 Detection of Mitochondrial Depolarization

The potentiometric fluorescent dye, JC-1, has a delocalized positive charge dispersed throughout its molecular structure. In addition, its lipophilic solubility enables it to be readily membrane permeant and penetrate living cells. Weakly positive charged JC-1 dye enters the negatively charged inner mitochondrial membrane matrix regions. As the dye accumulates, a critical concentration point will be reached leading to J-Aggregate formation. In contrast to the monomeric form of JC-1 which fluoresces green (527 nm) following blue light excitation (490 nm), JC-1 dye in the aggregated state (J-Aggregates) fluoresce orange (590 nm) upon blue light (490 nm) excitation. Healthy JC-1 stained cells, bearing proton-pump-functional mitochondria, concentrate JC-1 dye leading to J-Aggregate structure formation. The presence of J-Aggregate form-JC-1 within polarized mitochondria confers upon the recipient cell, the ability to fluoresce orange upon blue light excitation. Within cells bearing diminished or collapsed mitochondrial membrane potentials, which can occur during apoptotic or oxidative stress events, absence of a conducive environment for concentration and retention of positively charged JC-1 dye (J-Aggregate structure) leads to its conversion to the monomeric form of the dye. Monomeric forms of the JC-1 dye quickly equilibrate via simple concentration gradient action throughout the cytosol and into the extracellular media. This depolarization event is conveniently illustrated via a rapid drop in orange fluorescence staining properties of the target/experimental cell population. This leads to the default green fluorescence associated with remaining JC-1 monomer retained within the confines of the plasma membrane.

Alternative Potentiometric MitoPT Dye Options

ICT also offers two rhodamine-based mitochondrial depolarization detection probes. They include: TetraMethylRhodamineEthylester TMRE (Excitation/Emission: 549 nm / 574 nm) (2) and TetraMethylRhodamineMethylester TMRM (Excitation/Emission: 548 nm / 573 nm) (3). As was the case with JC-1, these rhodamine based dyes also fluoresce within the orange wavelength spectra. Their mechanism of action is synonymous to that observed with JC-1. TMRM and TMRE share physical properties common to all potentiometric fluorescent dyes. They possess a lipophilic molecular structure enabling them to penetrate both cell and mitochondrial membrane(s) barriers as well as a weak positive charge to facilitate their concentration within healthy polarized mitochondria. In contrast to the dual fluorescence properties of JC-1, TMRM and TMRE only emit at a single fixed wavelength within the orange spectrum (573 nm or 574 nm). Their selective concentration within healthy cell polarized mitochondria provides the biochemical basis for their utility as mitochondrial health status monitoring probes.

Successful Recent Science Using Several MitoPT Assay-Formats

The first papers describing this assay go all the way back to 2005 (4). Publications utilizing these techniques continue to appear at a steady pace. An example of some recent publications over this past year include: studies on treatment of Wilson Disease patients (5), bystander response on UV radiation (6), DKK3 function in hepatoma cells (7), and chemo-sensitivity of MCF-7 cells to a phytochemical with anti-cancer potential (8). Also during the past year, the MitoPT TMRE assay was used in publications for studies on platelet apoptosis in heart failure patients (9), on ferroptosis (10), and on Leber’s hereditary optic neuropathy (11).

References

1. Cossarizza, A., Baccarani-Contri, M., Kalashnikova, G. & Franceschi, C. A new method for the cytofluorimetric analysis of mitochondrial membrane potential using the J-aggregate forming lipophilic cation 5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethylbenzimidazolcarbocyanine iodide (JC-1). Biochem Biophys Res Commun 197, 40-45 (1993).
2. Yuan, X. M. et al. Lysosomal destabilization in p53-induced apoptosis. Proc Natl Acad Sci U S A 99, 6286-6291, doi:10.1073/pnas.092135599 (2002).
3. Heiskanen, K. M., Bhat, M. B., Wang, H. W., Ma, J. & Nieminen, A. L. Mitochondrial depolarization accompanies cytochrome c release during apoptosis in PC6 cells. J Biol Chem 274, 5654-5658 (1999).
4. Shih, P. H., Yeh, C. T. & Yen, G. C. Effects of anthocyanidin on the inhibition of proliferation and induction of apoptosis in human gastric adenocarcinoma cells. Food Chem Toxicol 43, 1557-1566, doi:10.1016/j.fct.2005.05.001 (2005).
5. Katerji, M. et al. Chemosensitivity of U251 Cells to the Co-treatment of D-Penicillamine and Copper: Possible Implications on Wilson Disease Patients. Front Mol Neurosci 10, 10, doi:10.3389/fnmol.2017.00010 (2017).
6. Le, M. et al. Exosomes are released by bystander cells exposed to radiation-induced biophoton signals: Reconciling the mechanisms mediating the bystander effect. PLoS One 12, e0173685, doi:10.1371/journal.pone.0173685 (2017).
7. Qui, S., Kano, J. & Noguchi, M. Dickkopf 3 attenuates xanthine dehydrogenase expression to prevent oxidative stress-induced apoptosis. Genes Cells 22, 406-417, doi:10.1111/gtc.12484 (2017).
8. Al Wafai, R. et al. Chemosensitivity of MCF-7 cells to eugenol: release of cytochrome-c and lactate dehydrogenase. Sci Rep 7, 43730, doi:10.1038/srep43730 (2017).
9. Mondal, N. K. et al. Mechanistic insight of platelet apoptosis leading to non-surgical bleeding among heart failure patients supported by continuous-flow left ventricular assist devices. Mol Cell Biochem, doi:10.1007/s11010-017-3021-1 (2017).
10. Neitemeier, S. et al. BID links ferroptosis to mitochondrial cell death pathways. Redox Biol 12, 558-570, doi:10.1016/j.redox.2017.03.007 (2017).
11. Carreno-Gago, L. et al. Identification and characterization of the novel point mutation m.3634A>G in the mitochondrial MT-ND1 gene associated with LHON syndrome. Biochim Biophys Acta 1863, 182-187, doi:10.1016/j.bbadis.2016.09.002 (2017).