I want to do mitochondrial membrane potential of human cell line. can anyone tell me the procedure of the assay? *We don't have flowcytometer in our lab.
Hi Deepa,
As others have noted, the rhodamine based fluorophores (TMRE/TMRM and Rhod-123) and the carbocyanins (JC-1 and DiOC6) are the most commonly used indicators of mitochondrial membrane potential and can be visualised using any suitable equipped fluorescent microscope. Care must be taken to choose the most appropriate indicator for your experiment and, as Andre Gomes points out, specific pharmacological controls should be employed to ensure results are interpreted correctly. Here are two links to the pdf versions of two open-access review articles discussing the advantages/disadvantages and correct usage of mitochondrial membrane potential probes (for transparency, I am a co-author on the second):
1 - http://www.biotechniques.com/multimedia/archive/00121/BTN_A_000113610_O_121224a.pdf
2 - http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3708117/?report=classic
Article Mitochondrial membrane potential probes and the proton gradi...
Hi Deepa,
There are some fluorescent dyes available which you can use for mitochondrial potential measurement. These dyes are viz. JC-1, TMRM, TMRE or many other rhodamine derivatives. You can culture your cells in the special dishes either 35/14 mm dishes or fluorodishes, which are required for microscopy. Once your cells are 80-90% confluent you can incubate them with any of the dye mentioned above along with mitotracker green - MTG (which binds selectively to mitochondria independent of potential and can be used to normalize the results). One important thing is that you can not use MTG with JC-1 as it is already a ratiometric probe and has two absorption maxima which produce both red and green fluorescence together (so in this case ratio of these two intensities gives you the potential). In my opinion TMRM/TMRE are the better probes. Once incubation is done, you can visualize these cells under fluorescence microscope and take several images. Once you have images you can do background subtraction and then you can easily calculate the ratio of TMRM/MTG fluorescence intensities which will be an indicator of the potential.
Hope this helps. Good luck!!!
Abhijit
JC-1 staining can be used. There also kits are available for this which are based on fluorimetry assays.. But you need a fluourescence reader. if you dont have it.. Then you can may be stain your cells with jc-1 and then quantitate the images using nih image j.. But this may not be the best way.
We use Dioc6 fluorescence and FCCP as a control for loss of potential. It can be done in a fluorescence microscope.
I used JC-1 before and it can be read both nder fluorescent plate reader or fluorecent microscope with adequate lens, or flow cytometry. However, some reviewers are extremely picky and will not accept this method. I have so many experience with reviewers insist one method is the gold standard whereas at the same time the other reviewer believe in the other way around. Invitrogen has a slew of the probes you can choose from. Good luck.
Hi Deepa,
As others have noted, the rhodamine based fluorophores (TMRE/TMRM and Rhod-123) and the carbocyanins (JC-1 and DiOC6) are the most commonly used indicators of mitochondrial membrane potential and can be visualised using any suitable equipped fluorescent microscope. Care must be taken to choose the most appropriate indicator for your experiment and, as Andre Gomes points out, specific pharmacological controls should be employed to ensure results are interpreted correctly. Here are two links to the pdf versions of two open-access review articles discussing the advantages/disadvantages and correct usage of mitochondrial membrane potential probes (for transparency, I am a co-author on the second):
1 - http://www.biotechniques.com/multimedia/archive/00121/BTN_A_000113610_O_121224a.pdf
2 - http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3708117/?report=classic
Article Mitochondrial membrane potential probes and the proton gradi...
- Badges
- Science method