Hi Experts,
I am trying to study the membrane potential of mitochondria of neuroglioma cells when exposed to a toxin. I tried using the Mitotracker Red CMXRos several times (from Life Technologies/Thermo Fisher) by a time course. But I don't get a substantial and "good" results that could meet my legitimate expectation. Has anyone used this type of dye for plate reader assays? Many protocols using this dye stain their cells or tissues after the treatment/intoxication, but not before. I also see a lot of dyes for mitochondrial staining:
1. DASPEI
2. DilC1(5)
3. JC-1 (chloride salt)
4. JC-1 (iodide salt)
5. JC-1 Mitochondrial Membrane Potential Detection Kit
6. MCB Glutathione Detection Kit
7. MitoView 633
8. MitoView Green
9. Nonyl Acridine Orange
10. Rhodamine 123
11. TMRE
12. TMRM
13. And those from Life Technologies Carbocyanine-based and Rosamine-based (i.e. Mitotracker Red CMXRos), which I used.
Is there a suitable dye for the type of cell and type of assay that I use and perform? Based on my initial findings, I might be able to use JC-1 or TMRE/TMRM dyes. Your opinion will be helpful.
Thanks for your help in advance.
Hello Edward,
measuring the mitochondrial membrane potential reliably is certainly no easy task. Many factors may influence this undertaking, especially culture conditions and cell type. I didn't work with mammalian cells so I can't share my experience with you. However, I found a publication that might be able to be useful for setting up your experiments (please see attachment). Good luck with your project!
Cheers, Christian
Article Quantitative measurement of mitochondrial membrane potential...
Have you looked at the number/intactness of mitochondria in your cell type? Some things to consider are that the mitochondria are sensitive to any ph, temperature, time changes. Also mitochondrial fission/fusion events occur very rapidly and the change in mitochondrial number may impact your results. I would supplement my mitotracker red cmxros assay with other membrane potential assays such as JC-1. Hope this helps!
Most of these dyes will work fine (TMRE and TMRM are especially fast) for microscopy applications because the membrane potential draws the dye into the mitochondria. However, since membrane potential just redistributes the dye, they won't work in a plate reader. You might try quantifying the JC-1 aggregate, but that might not be easy in a plate reader. Try to look at the mitochondria directly in a fluorescence microscope.
Hi Christian,
Thanks for your response and attached file!
Cheers,
Edward
Hi Jamila,
Thanks for your response. I am trying to optimize our conditions and assay protocols for the type of cell that we have. Could you provide any reference to follow? By the way thanks for pointing out some factors that can affect mitochondrial integrity and function. I will definitely take note of that. Right now, I am trying to determine the effects of a toxin on mitochondrial membrane potential in real time using a plate reader detecting its fluorescence activity. Preliminary experiments that will make my experiment go forward and probably expand based on what I will get incorporating your suggestion. I actually used the Mitotracker Red CMXROS for a couple of times and my data doesn't look that great. Even control shows erratic and unsubstantial results. I might try TMRE or TMRM, but I am wondering which one of these is applicable to my experiment.
Thanks,
Edward
Hello Eduardo,
For the mitochondrial potential, the most of the mitrotracker dyes work well, also Rhodamine 123 is a very good dye.
- Thanks. According to one link below, TMRE or TMRM might be the best for live cell assay of mitochondrial membrane potential.
Probably you need to include in your protocol the appropriate controls. For example, after measurement of the potential, treat your cells with FCCP in order to disrupt the potential.
- I am using proper controls, but every time I do it, results from control are inconsistent. I am treating my cells with toxins and determining its effect on membrane potential. So maybe Mitotracker Red CMXRos is not the best fit for my assay.
Thanks for your response!
https://www.thermofisher.com/us/en/home/references/molecular-probes-the-handbook/probes-for-organelles/probes-for-mitochondria.html#head7
Hi Alexander,
Most of these dyes will work fine (TMRE and TMRM are especially fast) for microscopy applications because the membrane potential draws the dye into the mitochondria. However, since membrane potential just redistributes the dye, they won't work in a plate reader.
- Hmm, it should be alright. Does the dye fluoresce when unbound to membrane?
You might try quantifying the JC-1 aggregate, but that might not be easy in a plate reader. Try to look at the mitochondria directly in a fluorescence microscope.
- I read some papers, JC-1 works with 2 fluorescence FITC and TRITC. The problem there is time delay between changing fluorescence. I think TMRE and TMRM is the best fit for my assay. And I always look at my cells under the microscope. Also see the link I included here. Hope this information is also helpful!
https://www.thermofisher.com/us/en/home/references/molecular-probes-the-handbook/probes-for-organelles/probes-for-mitochondria.html#head7
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