Use manitol purified mitochondria for JC1. It helps to get rid of unwanted things and gives pure mitochondria.

Hi Michael,

I attached a protocol for the use of JC1-stained mitochondria in flow cytometry. Maybe it is useful to you. Good luck!

Best regards, Christian

Sorry, the protocol I posted is for use with mitochondria in cells... Will let you know if I find information on how to analyze isolated JC1-labeled mitochondria with FACS.

Do not use JC1 at all. Look for something better depending on your purpose.

Hi Gentlemen,

Thank you for the responses and I will address each in turn.

Goodwin- I'm not so concerned about the debris as the technique I use is well developed and widely used within our lab and others. Further, I have also used the isolated mitochondria on live assays on the Seahorse and so the quality of the isolates aren't an issue for me. The debris is found in any buffer I run, PBS, HBSS or mitochondrial isolation buffer (all of which my mitochondria could be resuspended in depending upon the downstream assays). Based upon the spread of events in the FSC and SSC scale with a proportion of the mitochondria located/overlapping the area populated by debris presenting in the buffer only sample initially run

Christian- thanks for your help, I have had no luck finding this information to date, so if you can find anything I would be most grateful.

Alexander- my purpose is to identify membrane potential in isolates from kidneys of treated animals and so mitochondrial isolations I thought were my only possibility. These have all been freshly isolated and frozen before one thaw cycle and analysis. I am viewing differences between groups, but was hoping to gain information on others who may have insight in using this technique previously.

One more thank you all for taking the time to offer help and advice in the process. This is an invaluable resource for one and all!

Micheal

With the isolated mitochondria the best and most reliable method is to use a TPP-sensitive electrode, which is easy to make. Though, it requires some instrumentation, But it worth it.

I've measured membrane potential in isolated mitochondria with JC-1 by both fluorescence reader and microscopy, and it worked well.

For flow citometry I found for example this article:

Cossarizza, A., Baccarani-Contri, M., Kalashnikova, G., and Franceschi, C. (1993) 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). Biochemical and biophysical research communications 197: 40-45.

Good luck!

Alicia

http://www.ncbi.nlm.nih.gov/pubmed/8250945

Hi Ladies,

Thank you for the advise it is much appreciated. Alicia, unfortunately as with quite a number of articles available on JC-1 use this article again is focused on cells rather than isolated mitochondria, but thank you for your help on the subject!

Micheal

Michael, I can relate. I, too, am trying to stain purified mitochondria with JC-1 and getting some very strange results. I get very little staining in the red and green channels at all, and a huge amount of unstained material. Not sure if my mitochondria are dead or if it is a JC-1 problem.

I wonder if anyone have found a protocol for Mitotracker staining isoalted mitos from mouse tissue ( not cells) ?

Dear Alicia Virseda Jerez,

thank you for the reference. I have found the paper, and, as I have expected, the authors used culture medium with Pen/Strep. This is a very good demonstration of why I recommended not to use JC1 at all. The problem with this die is that it sticks to anything. In cells grown in the presence of streptomycin mitochondria do not function normally. They are alive, they will respond to changing situation in the cell, but they do not respire, they have no oxidative phosphorylation. I have many times different cells grown without and with Pen/Strep, and in all cases isolated mitochondria had no respiration. This is a worldwide tragedy, that people working with cells use Pen/Strep and think they do Science. Douglas C. Wallace, the Father of mitochondrial Medicine, every week used to warn us do not use antibiotics! Mitochondria are former bacteria< therefore, they are sensitive to antibiotics. I (that is Dug) instantly fire anyone, who uses antibiotics. I was lucky that I studied cell culturing in his lab. My warned my friends, who use cell culturing< said to me that after they stopped using Pen/Strep, they started to obtain absolutely different results that make sense. Do not waste your time and Life doing nonsense.

I guess it is good idea to be aware of potential limitations of experimental approach. At the same time to keep moving forward scientists have to try any potential solutions with appropriate controls of cause.

@livia : I have optimized the protocol for mitotracker staining for isolated mitochondria. And i was getting good results.

But i am further confirming results.

To Micheal S Ward , sorry to respond to your above notion. You mentioned that for evaluation you use frozen mitochondria. This is entirely wrong thing! Your mitochondria after thawing become damaged. Isolation of kidney mitochondria described in my "Practical mitochondria" gives high quality and you can use them for at least 4-5 hours. Recently we have found that kidney mitochondria have several functional differences from heart, brain or skeletal muscle mitochondria. Basically, they have no or only weak endogenous inhibition of succinate dehydrogenase. But still, the best substrates are 25 mcM palmitoyl-L-carnitine + (glutamate 5 mM+ malate 2 mM) or succinate 5 mM. These are physiological mixtures of substrates. In no case use succinate+rotenone (these days it is regarded as a "stupid" mixture).