JC-1

JC-1 is a dual-emission potential-sensitive probe that can be used to measure mitochondrial membrane potential. JC-1 is a green-fluorescent (λex 520 nm) monomer at low membrane potential. At higher potentials, JC-1 forms red-fluorescent (λem 596 nm ) "J-aggregates," which exhibit broad excitation and very narrow emission spectra. The ratio of red to green fluorescence of JC-1 is dependent only on membrane potential, and not influenced by mitochondrial size, shape, or density. JC-1 is particularly useful for apoptosis studies. In apoptotic cells, the dye stays in the cytoplasm and fluoresces green, while in healthy cells, the dye aggregates in the mitochondria and fluoresces red.

Ratiometric high-resolution imaging of JC-1 fluorescence reveals the subcellular heterogeneity of astrocytic mitochondria.[Pubmed:21881871]

Pflugers Arch. 2011 Nov;462(5):693-708.

Using the mitochondrial potential (DeltaPsi(m)) marker JC-1 (5,5',6,6'-tetrachloro-1,1',3,3'-tetraethylbenzimidazolylcarbocyanine iodide) and high-resolution imaging, we functionally analyzed mitochondria in cultured rat hippocampal astrocytes. Ratiometric detection of JC-1 fluorescence identified mitochondria with high and low DeltaPsi(m). Mitochondrial density was highest in the perinuclear region, whereas DeltaPsi(m) tended to be higher in peripheral mitochondria. Spontaneous DeltaPsi(m) fluctuations, representing episodes of increased energization, appeared in individual mitochondria or synchronized in mitochondrial clusters. They continued upon withdrawal of extracellular Ca(2+), but were antagonized by dantrolene or 2-aminoethoxydiphenylborate (2-APB). Fluo-3 imaging revealed local cytosolic Ca(2+) transients with similar kinetics that also were depressed by dantrolene and 2-APB. Massive cellular Ca(2+) load or metabolic impairment abolished DeltaPsi(m) fluctuations, occasionally evoking heterogeneous mitochondrial depolarizations. The detected diversity and DeltaPsi(m) heterogeneity of mitochondria confirms that even in less structurally polarized cells, such as astrocytes, specialized mitochondrial subpopulations coexist. We conclude that DeltaPsi(m) fluctuations are an indication of mitochondrial viability and are triggered by local Ca(2+) release from the endoplasmic reticulum. This spatially confined organelle crosstalk contributes to the functional heterogeneity of mitochondria and may serve to adapt the metabolism of glial cells to the activity and metabolic demand of complex neuronal networks. The established ratiometric JC-1 imaging-especially combined with two-photon microscopy-enables quantitative functional analyses of individual mitochondria as well as the comparison of mitochondrial heterogeneity in different preparations and/or treatment conditions.

Lenticular mitoprotection. Part A: Monitoring mitochondrial depolarization with JC-1 and artifactual fluorescence by the glycogen synthase kinase-3beta inhibitor, SB216763.[Pubmed:23825920]

Mol Vis. 2013 Jun 27;19:1406-12. Print 2013.

PURPOSE: Dissipation of the electrochemical gradient across the inner mitochondrial membrane results in mitochondrial membrane permeability transition (mMPT), a potential early marker for the onset of apoptosis. In this study, we demonstrate a role for glycogen synthase kinase-3beta (GSK-3beta) in regulating mMPT. Using direct inhibition of GSK-3beta with the GSK-3beta inhibitor SB216763, mitochondria may be prevented from depolarizing (hereafter referred to as mitoprotection). Cells treated with SB216763 showed an artifact of fluorescence similar to the green emission spectrum of the JC-1 dye. We demonstrate the novel use of spectral deconvolution to negate the interfering contributing fluorescence by SB216763, thus allowing an unfettered analysis of the JC-1 dye to determine the mitochondrial membrane potential. METHODS: Secondary cultures of virally transfected human lens epithelial cells (HLE-B3) were exposed to acute hypoxic conditions (approximately 1% O(2)) followed by exposure to atmospheric oxygen (approximately 21% O(2)). The fluorescent dye JC-1 was used to monitor the extent of mitochondrial depolarization upon exposure of inhibitor treatment relative to the control cells (mock inhibition) in atmospheric oxygen. Annexin V-fluorescein isothiocyanate/propidium iodide staining was implemented to determine cell viability. RESULTS: Treatment of HLE-B3 cells with SB216763 (12 microM), when challenged by oxidative stress, suppressed mitochondrial depolarization relative to control cells as demonstrated with JC-1 fluorescent dye analysis. Neither the control nor the SB216763-treated HLE-B3 cells tested positive with annexin V-fluorescein isothiocyanate/propidium iodide staining under the conditions of the experiment. CONCLUSIONS: Inhibition of GSK-3beta activity by SB216763 blocked mMPT relative to the slow but consistent depolarization observed with the control cells. We conclude that inhibition of GSK-3beta activity by the GSK-3beta inhibitor SB216763 provides positive protection against mitochondrial depolarization.

Uncompensated Polychromatic Analysis of Mitochondrial Membrane Potential Using JC-1 and Multilaser Excitation.[Pubmed:25827483]

Curr Protoc Cytom. 2015 Apr 1;72:7.32.1-11.

The lipophilic cation JC-1 (5,5',6,6'-tetrachloro-1,1',3,3'-tetraethyl-benzimidazolyl carbocyanine iodide) has been used for more than 20 years as a specific dye for measuring mitochondrial membrane potential (DeltaPsi(m)). In this unit, we revise our original protocol (that made use of a single 488 nm laser for the detection of monomers and aggregates, and where compensation was an important step) to use dual-laser excitation. Moreover, thanks to recently developed multilaser instruments and novel probes for surface and intracellular markers, JC-1 can be utilized by polychromatic flow cytometry to simultaneously detect, without any compensation between fluorescences, DeltaPsi(m) along with other biological parameters, such as apoptosis and the production of reactive oxygen species.

JC-1: alternative excitation wavelengths facilitate mitochondrial membrane potential cytometry.[Pubmed:23171850]

Cell Death Dis. 2012 Nov 22;3:e430.

Mitochondrial membrane potential provides a valuable indicator of cells' health and functional status. Cytometry- and microscopy-based analyses, in combination with fluorescent probes, are widely used to study mitochondrial behavior related to cellular pathways, most notably - apoptosis. The cyanine dye JC-1 (5,5',6,6'-tetrachloro-1,1',3,3'-tetraethylbenzimi- dazolylcarbocyanine iodide) facilitates discrimination of energized and deenergized mitochondria because the normally green fluorescent dye forms red fluorescent aggregates when concentrated in energized mitochondria in response to their higher membrane potential. JC-1 fluorescence is usually excited by the 488 nm laser wavelength common in flow cytometers. In this study, we show that in practice this approach is not optimal for monitoring mitochondrial behavior. Investigation of fluorescence of JC-1 in solution and in cells using spectrofluorimetry, microscopy and flow cytometry reveals that excitation at 405 nm wavelength, now available on standard instruments, produces signals from aggregate fluorescence with considerably less spillover from dye monomer fluorescence than can be obtained using 488 nm excitation. The improved data are more accurate and eliminate the necessity for fluorescence compensation, making the use of the alternative excitation wavelengths beneficial for mitochondria-related biological and biomedial research.