I suggest TMRM, as some previous poster also did. The crucial bit is to ensure not to use such cationic dYm dyes in quench mode during imaging. In our hands, typically 30 nM is sufficient. Also note that these dyes are sensitive to changes in dYp as well, which may need to be considered depending on your setting. The work by David Nicholls is very authoritative in this respect. I added a few citations below that may help you for your work. TMRM definitely outperforms mitotracker for time lapse imaging. Works great for FACS as well. Careful with DAPI co-stains and similar, since these dyes are fairly toxic, especially when doing time-lapse imaging.

http://www.jneurosci.org/content/20/19/7208.long

http://jcs.biologists.org/cgi/pmidlookup?view=long&pmid=12508113

Hi Shakti ranjan, you can look this paper, they have done some sort of mito membrane potential measurement through microscopy as well as some biochemical experiments. http://www.ncbi.nlm.nih.gov/pubmed/23720737.

for nucleus staining you can try hoechst, it will stain DNA.

good luck,

Regards,

Ashvini Kumar Dubey

We used a fluorescent dye to measure and monitor mito membrane potential. It is described in one of our paper.

Cell Death Differ. 2013 Mar;20(3):478-89. doi: 10.1038/cdd.2012.144. Epub 2012 Nov 23.

T-cell death following immune activation is mediated by mitochondria-localized SARM.

There are other dyes that can be used to measure MMP, like TMRM or JC1.

They all work well, at least for flow cytometry (FACS). You can check our paper: http://www.ncbi.nlm.nih.gov/pubmed/20037159

Here we used TMRM to measure MMP by FACS.

I do believe the choice of dye is not critical here. Mitotracker should also work well for microscopy. I guess the problem is that it is hard to get a good quantitave result by using microscopy. You need to take many pictures and use an imaging analysis software (such as ImageJ) to quantify the results. If you take enough images from several independent experiments, comparing you treatment to a control group, you might be able to get a quantitative and statistically significant difference between the groups.

Also, it might be worth trying to use a more quantitative method like FACS for your experiments. If you can get it working it will make your life much easier :-)

Let me know if you have any question.

Dear Dmitri

Thanks for the suggestions. I have performed the MMP assay with mitotracker dye. It really worked well. I have checked for several drugs and it all gave promising results. But I am trying to do a microscopy based MMP assay with Mitotracker. I did twice but didnt find satisfactory results compared to FACS. Please suggest any protocol regarding that.

Thanking you.

What is the problem that you are specifically running in to with your staining? Is your staining inconsistent? Faded? Also are you using LSCM, SDCM, Widefield, or a regular scope with an epifluorescent light source? Are you using any deconvolution software to process your images?

Dear Anthony

I am facing problem while staining with Mitotracker dye and nucleus with DAPI staining. I am not finding good results while merging the both. I do image processing with ImageJ software. Please suggest me a protocol for better staining and microscopy.

Thank you.

Shakti

I have used mitotracker orange, which is retained well after formaldehyde fixation, and then stained the nuclei with hoechst. What is your analysis output for the mitotracker? Spot counting, fluorescence intensity? I have used spot counting and texture analysis to quantitate MMP. You will have to optimize your staining concentration and treatment time points to optimize the assay and subsequent image analysis.

Now there are a couple things to this, first off, Angie used MitoTracker Orange which I believe is a Rosamine-based dye. Some MitoTracker Reds are Rosamine-based as well, but if it is a Carbocyanine-based dye, then it may not retain well after fixation (meaning it should be used in live cells). Another thing is to not stain fixed cells, but perform the fixation after your incubation with the MitoTracker dye.

As Angie stated, optimizing your treatment concentration and incubation time need to be adjusted based on whether your staining is faint (increase concentration/time) or you have too much background (decrease the concentration/ increase time).

For DAPI staining, are you using a DAPI solution that you add for a few minutes or a DAPI mounting medium? Also another thing to consider is possible photo-bleaching. Are you using a mounting medium with anti-fade? I used to make my own using 0.5% N-propyl gallate, 90% Glycerol , 20mM Tris pH 8.0.

Also, if you don't know how to overlay images in ImageJ, try reading about channels and merging them here: http://rsbweb.nih.gov/ij/docs/menus/image.html#color.

I suggest TMRM, as some previous poster also did. The crucial bit is to ensure not to use such cationic dYm dyes in quench mode during imaging. In our hands, typically 30 nM is sufficient. Also note that these dyes are sensitive to changes in dYp as well, which may need to be considered depending on your setting. The work by David Nicholls is very authoritative in this respect. I added a few citations below that may help you for your work. TMRM definitely outperforms mitotracker for time lapse imaging. Works great for FACS as well. Careful with DAPI co-stains and similar, since these dyes are fairly toxic, especially when doing time-lapse imaging.

http://www.jneurosci.org/content/20/19/7208.long

http://jcs.biologists.org/cgi/pmidlookup?view=long&pmid=12508113

Shakti:using JC-1 is a good suggestion. IT has a reddish jue and turns green when the membrane potential changes. This can be picked up by epifluorescence. There are good review articles out there on this topic and what dye to use, but JC-1 does the job. Look up Perry et al 2011 in BioTechniques. We us it in adipocytes after forskolin treatment. Regarding the nucleus, DAPI et al work only on dead=fixed cells and you want to keep cells alive for your purposes. Perhaps you have the option after the assay is doen to just switch to phase contrast and thus capture the nucleus. Any dye that stains the nucleus intercalates with DNA and I would be careful not to have other dyes roaming the cytoplasm if you really want to make sure you want to keep other organelles untouche. Best wishes, Michaele Ragnunath , Dept of Biomedical Engineering, National U of Singapore.

Thank you sir for you much valuable suggestion and guidance. I read the paper and found it much informative.

Best Regards

Shakti