You can use JC-1 for flow cytometry or spectrofluorimetric analisys - but the data interpretation is the same: The excitation wavelength for JC-1 is 490 nm and the emission wavelengths are observed at 535 nm (fluorescence characterised for JC-1 monomers) and at 595 nm (fluorescence characterized for JC-1 aggregates). Then the ratio of monomers to aggregates (535/595) is evaluated. The higher value of this ratio the higher membrane depolaryzation will be observed. When the potential of mitochondria is high, JC-1 is moved inside mito and the aggegates of JC-1 are formed, while during low mito potential, monomeres of JC-1 are formed. Aggregates are formed inside of mito because there is highly negative mito matrix and the potential is enough high to "pull" the label. Summary, when mito are in a good conditions, they work properly their mitochondrial potential is high and then the entrance of JC-1 is fast, but during mito damages, the potential is lower, it means that the mitochondrial inner membrane becomes less negative (much more positive) and the force needed to pull of this label is weaker. Total potential for whole mitochondria is not the same - therefore in mitochondria, JC-1 CAN FORMS AGGREGATES AS WELL AS MONOMERS - we can observe both of them - therefore using JC-1 we show results as a ratio of monomers to aggregates - the higher ratio the mito depolarization higher.

The equilibrium between JC-1 monomers and dimers is concetration dependent. Upon addition of JC-1 stock to a cell suspension it has to dissociate and by dilution monomers will be formed which enter the cytosol. From there the mitochondria accumulate JC-1 forming dimers in the matrix. Upon disruption of the membrane potential JC-1 will be diluted into the cytosol and dimers inside the matrix disappear.

Answer A: Not whole JC-1 will got into intact mitochondria, but a high portion

Answer B: Yes

You can use JC-1 for flow cytometry or spectrofluorimetric analisys - but the data interpretation is the same: The excitation wavelength for JC-1 is 490 nm and the emission wavelengths are observed at 535 nm (fluorescence characterised for JC-1 monomers) and at 595 nm (fluorescence characterized for JC-1 aggregates). Then the ratio of monomers to aggregates (535/595) is evaluated. The higher value of this ratio the higher membrane depolaryzation will be observed. When the potential of mitochondria is high, JC-1 is moved inside mito and the aggegates of JC-1 are formed, while during low mito potential, monomeres of JC-1 are formed. Aggregates are formed inside of mito because there is highly negative mito matrix and the potential is enough high to "pull" the label. Summary, when mito are in a good conditions, they work properly their mitochondrial potential is high and then the entrance of JC-1 is fast, but during mito damages, the potential is lower, it means that the mitochondrial inner membrane becomes less negative (much more positive) and the force needed to pull of this label is weaker. Total potential for whole mitochondria is not the same - therefore in mitochondria, JC-1 CAN FORMS AGGREGATES AS WELL AS MONOMERS - we can observe both of them - therefore using JC-1 we show results as a ratio of monomers to aggregates - the higher ratio the mito depolarization higher.

You have the answer already in two good replies. But some detail. Assuming -30 mV across plasma membrane and -120 mV across mito membrane, 10 nM JC-1 in medium would equilibrate with 32 nM in the cytoplasm (showing green fluoresence), and 3,200 nM in the matrix. At that concentration the dimers form and give red fluoresence. But equilibrium is slow at that low concentration. Most workers add 10 uM (red fluoresence) externally for 10 minutes, then wash away the external, but that seems to me somewhat 'hit-or-miss'.

in different laboratories, people use different JC-1 concentration and time -incubation with the label differs too. Probably it depends on the used material (culture cells, tissue etc), used apparatus etc.... . therefore it is very important to have so called 'positive control' to be sure that we do not pool artifacts :)

Just to close the circle a bit, JC-1 goes into mitochondria because it is positively charged. The somewhat complex nature of what one measures experimentally (local concentration dependent aggregation) means one has to be very careful about employing consistent conditions and using appropriate controls. Whether JC-1 is the dye of choice depends on the experiment. Some bioenergenicists avoid it altogether in favor of "true" potentiometric dyes such as TMRE.

The JC-1 dye is cationic and lipophilic. so, it goes to the more electronegative sink, the mitochondria, with a concentration increase by approximately X1000 compare to the ouside of the cell provided you can estimate - 60 mV across the plasma membrane and around -120 mV across the mitochondrial membrane ... Since if you have 10 nm outside you will reach 10 microM inside mitochondria, if the mitochondria maintained their potential. Less potential means less dye accumulated and a drop of potential means a release of the dye within the cytoplasm. But this is not the easiest dye to use. Depending of the cell characteristic, I will prefer DiOC6(3) at 10 to 40 nM outside, or TMRM/ TMRE or Deep red (5-10 nM outside)... which are more potents... membrane potential dye indicators

Always try to uncouple your mitochondria for a negative control.

Their is always an amount of JC-1 in the cytoplasm if the plasma membrane potential is maintained... But the concentration is negligible... compare to the concentration within the mitochondria. If you uncouple with FCCP (do a titration on your cells because the concentration to be used may vary) and you also uncouple the plasma membrane potential, then you have the proper control where almotst no dye can penerate neither the cytoplasm nor the mitochondria...

Patrice

NB 1 the red/green fluorescence is somehow difficult to follow for example in apoptosis, since the cytoplasmic pH is changing and the green fluorescence of JC-1 is more or less sensitive to pH change.

Better use another dye and always do some viability assay (double staining) for safety

NB 2 for uncouplig you ca also use a couple of nigericin (for delta pH) and valinomycin (for the potential) (with a mediun containing KCl).

Also, the preparation of the dye JC-1 is some how difficult, you may avoid aggregates (so, the concentration you use may be affected by that)...Effectively TMRM or TMRE are better potentiometric dyes... And aloso useful for imaging.

I started to love Deep red also! Very good probe for imaging which let other channels free!

JC-1dye preparation is really cumbersome and data explanation is also. Dioc6,TMRM is good but, TMRM is really unspecific, Dioc6 concentration selection is difficult, Rh123 uptake is different in different cells, so the inference of MMP. Dyes are like that only, getting information is an art.....science is mysterious..

I am not sure Krish is fully rigth...

The answer is a bit short.

Bioenergetic comprehension is needed to use these dyes...

But anyhow, sit by your cells and try seriously.. with control and screening of different concentration avoiding artefacts; pH changes, dye quenching, fluorescence life time, photoactivation etc... You need also a good comprehension of the cellular metabolism.

Good luck indeed.. That why we are research scientists and not only technicians!!!

Litterature is needed, even if full of unwanted dirty publications...

Patrice

By the way , Hi! Carmen still here, its a pleasure...

Patrice

Hi Patrice! Still here also. Your last answer is the ultimate correct answer. Make a best guess at what dye would work best for your experiment, then study your system behavior carefully before trusting it. Always do controls, know the literature and seek advice of experts like you. Cheers!

To Ian west...

If people use 10 microM outside, they will reach a situation of high quenching of the probe inside mitochondria, and also a huge cytotoxicity toward the fundamental mechanisms under study, i.e. the mitochondrial respiratory chain and the potential they are supposed to mesure. Since the I50 of the FoF1 ATPase is at 125 microM... all the mitochondrial bioenergetic are perturbed... Moreover they will generated artefacts which are laying into the many publication for years (this is particularly true for Rh123), since an initial drop in potential will dequenche the fluorochrome leading to fluorescence increase (they will then estimate that their potential increase when it has already decrease...) and after an important drop and later on they will say the effectively their fluorescence is decreasing... when working properly they will just find that a decrease of fluorescence correspond to a decrease of potential.... except in very rare situations where the cells counteract the stress by a transient increase of mitochondrial membrane potential).. But, if you find this type of publications tell me because they are very exceptional.

Thank you all . Really this is very much nice information regarding JC-1 action.

I prefer TMRE, which is sensitive and easy to analyze. I am not sure about Krishnanand Mishra's comment about TMRM being 'really unspecific'. When using TMRE should use only low concentration- 50 nM. Some simply dump the TMRE dye, such as 100 nM and above, which really is unspecific. Ratiometric dyes are problematic and discussed in many publications previously, including mine. Some ratiometric dyes are toxic to cells. As Patrice Petit mentioned, one should always use positive and negative controls.