I want to measure the Mitochondrial Membrane Potential using fluorescence dyes e.g. JC-1, TMRE. My experimental setup is at 24-Wellplate (not changeable). I want to incubate the cells as described by the manufactures protocol. However, as our fluorescence microscope is to old, I want to lyse the cells after incubation with the dye and transfer it to a 96-Well plate to measure it in a micro plate reader. Has anybody experience or a protocol what dye is stable (as they from aggregates in the mitochondria) and for how long. I was thinking that works similar to MTT, where you also have an accumulation of formazan and lyse the cells and measure in the cell lysate. As I am expecting that my treatment increases the MMP, would JC-1 be the better choice then? (Is my assumption correct that TMRE is e.g. to show MMP decreases compared to control or can i also detect and increase in MMP compared to untreated control?) Thank you very much beforehand!
Hi Janine,
How are you planning on lysing the cells? If your method of lysis disrupts the cell membrane, you always run the risk of disrupting the mitochondrial membranes. All of the aforementioned dyes are cationic and will accumulate in the matrix based on charge - that being said, if your mitochondria become depolarized during lysis step, you're likely to lose your membrane potential indicators as well. TMRE is a relatively bright probe and can be measured on a fluorescent plate reader in live cells - and chance this might work with your scope, or have you considered a plate reader as an option?
if you go the TMRE route, please be sure to use it in the 10-20 nM range - any higher and the dye can quench itself.
Best of luck!
Hi, Janine.
Yes. So right.
Please keep away from light.
Read the results within half an hour.
Excitation wavelength: 488nm
Emission wavelength: 520nm
TMRM, TMRE, and other Rh123 derivatives as well as JC1 are suitable for live imaging. As soon as mitochondria looses membrane potential (caused by lysis for instance) all the dies are going to escape from mitochondria.
For microscopy TMRM (TMRE) are used in so-called not quenching mode (5-100 nM) but for plate reader this mode is not suitable because total TMRM fluorescence (signal you detect from whole well) is independent of the dye compartmentalization (inside mitochondria or in the cytosol).
For platereader you should use TMRM (TMRE) in quenching mode (say 0.5-2 uM), the mode used for isolated mitochondria. In this case accumulation of TMRM in mitochondria results in self-quenching of the fluorescence so higher membrane potential would correspond to lower TMRM fluorescence intensity.
Keep in mind that other parameters like protein concentration, or cell count, or mitochondria count, or TMRM toxicity at high concentrations etc should be controlled. Therefore I doubt you can use it for cells.
As a suggestion, you can try to incubate your cells with Mitotracker the fixable one (say MitoTracker Deep Red). Then fix and wash the cells and simply measure Mitotracker fluorescence yield. Indeed it is going to be challenging to ensure that all the controls are done correctly.
Good luck!
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