Concerns with JC-1 are outlined here: http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3115691/

in short...JC1 is ratiometric (if you have the 2 lasers) while TMRE is not. So it means that JC1 is more realiable (in the ratiometric way of analysis), and in my experience, also less "dirty" (almost no spurious binding outside the mitochondria). that is ..good luck

I confirm above information about JC-1. This fluorescent lable works very well in cytometr analysis as well as in spectrofluorimetic measurements. Nevertheless, it is a good practise to read more about these probes (JC-1 and TMRE) before experiments to know advantages and disadvantages of each of them. If you have this possibility, try to use both of these probes :)

Where as TMRE is monochromatic JC-1 is ratiometric probe. The latter produces a green fluorescence in its monomeric form which then aggregates in mitochondria to induce a spectral shift to Red. Both dyes are semiquantitative but JC-1 can be less accurate. This is due to a number of reasons such as time such as rate of change in green vs red fluorescence and the sensitivity of JC-1 towards changes in hydrogen peroxide. The former point is important since equilibration of the monomeric green signal only takes 15 min (similar to TMRE or TMRM) whereas equilibration of the aggregate can take up to 90 min. Essentially this means that the JC-1 probe can provide false positives in terms of measurement of membrane potential.

Thus, TMRE or TMRM or for isolated mitochondria Safranin O, are typically used for more sensitive measurements time dependent measurements in membrane potential whereas JC-1 is more suitable for end-point analysis.

very valuable information from Ryan :) I agree with him in 100%. It is very important to stress that in theory the mechanism of JC-1activity is esasy and reliable but in practise - not always it is so obvious. I know people who use JC-9 and they are very glad from this 'changing" :)

Thanks a lot is Martin and Ryan for such valuable information. I am dealing with this issue with my drosophila mitochondria.

I use Mitotracker orange.

In my hands, JC-1 works pretty well... and i agree with Ryan poster that JC-1 is best suits for end-point analysis.

Mitotracker and JC-1 both are good. For both you should perform additional essay to identify possible factors contributing to the results observed.

JC-1 is more specific to mitochondrial membrane potential, whereas TMRE will respond to changes in both, mitochondrial and plasma membrane changes.

JC1 unfortunately leads to artefacts. To date the best probes are TMRM or TMRE. If you use the confocal intact cells you should take in account also the plasma membrane potential

What kind of artefacts Marco Spinazzi?

Both TMRE and JC1 dyes are lipophilic cations which accumulate in mitochondria according to the Nernst equation. JC-1 is slightly different in that aggregation causes a shift in fluorescence from green to red, with the ratio between the two signals giving a measurement of Δψm. Particular care must be taken when interpreting results derived from JC-1 measurements as the red aggregated signal has been reported to be influenced by factors other than Δψm. So my experience with TMRE or TMRM was better. See Parihar et al., Biochimica et Biophysica Acta 1780 (2008) 921–926; The International Journal of Biochemistry & Cell Biology 41 (2009) 2015–2024; Journal of Neuroscience Research 85:1018–1032 (2007)

JC1 aggregation has been reported to depend also from factors independent from Dpsi and, most importantly, it inhibits mitochondrial respirations (which will affect the Dpsi)

Marco, do you have any papers, may reviews, confirming the opinions of JC-1 activity you i ntroduced above?

Agree with all of the above. The experimental design should also be considered.

As Ryan pointed out, JC-1 is better suited for end-point measurements, given the time it takes to equilibrate between non-aggregated and aggregated forms. In addition, in my hands this process was not reversible, at least not in the dynamic sense that would allow time-resolved measurements.

I have been able to perform time-resolved imaging with TMRE where cells were stained prior to treatment and I measured the decrease in fluorescence intensity over time. The advantage of the latter is that your results are expressed as rates or ratios or fluorescence at time=t over time=0, therefore each sample serves as its own control and variances in staining efficiency can be rooted out this way.

I am in agreement with Sebastien Boridy.

Our lab uses JC-1 but not other reagents for endpoint measurements of inner mitochondrial membrane potential. I tried to adapt JC-1 for a real-time measurements, which the company (Cayman) said was "theoretically possible". I was not able to do so, however I also work with mixed human-bacterial cell samples, so I suspect that the bacteria in the sample took up some JC-1 reagent (due to their similarities in membrane structure to mitochondria). The appropriate controls supported this. Because this was my end goal, I did not look into human cell-only samples, though the company (Cayman) does say it's possible.

Sebastien, if I go back to do this experiment again, I will try with TMRE due to your real-time success with it. However, it doesn't change the caveat for my research of working with mixed human cell-bacterial cell samples, too. Appreciated!

I recommend that, no matter if you use JC-1 or TMRE/TMRM, to use an uncoupler such as FCCP, CCCP, or DNP as control. This will verify that what you measure is truly mitochondrial membrane potential.

JC-1 is easier to get usable data with the least optimization, especially if your experiment is cell culture and you're using a plate reader to detect red vs green. If you have easy access to flow cytometer or fluorescence microscope, I'd recommend TMRE/TMRM. See John Lemaster's papers using Mitotracker Green and TMRM as one idea to visualize both mitochondria and membrane potential. Lastly, TMRM/TMRE with microscopy allows you to calculate membrane potential (also see Lemaster's papers), whereas JC-1 is very qualitative (more vs less than control group).

I agree that TMRM or TMRE would be better probe for measuring membrane potential using FCCP to depolarise at the end.

Has anyone used either of these dyes with platelets?

We have not tried JC-1 so far, but we use other fluorescence probes in conjuntion with respirometry and observed considerable toxicity of safranin and also TMRM at higher concentrations (which however are often used). However, up to 2 µM TMRM is apparently not toxic. You may not see the toxic effects without simultaneous respirometry, but that does not mean they are not there then.

For reference please see:

Scaduto et al. Biophys J 1999: 469–477.

Krumschnabel et al. Methods Enzymol 2014: 163-81.

Di-8-Anepps is one of the best probes you could use. You can even calibrate the probe to determine the membrane potential. It is a ratiometric probe. We have used that one for years...

I know Di-8-Anepps is great for cell membrane potential; can it be localized to the mitochondria?

Ah, I see, it was about Mitochondria... I just read the title topic and wondered why you're talking about JC1 and "changes in membrane potential". There is actually a nice paper on " Mitochondria specific VSDs". http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3115691/