I would like to measure mitochondrial mass, and wanted to use the Nonyl Acridine Orange (Acridine Orange 10-Nonyl Bromide).
I have used the MitoTracker Green for measurement, although it might be independent to membrane potential, it seems to be interacting with the uncouplers like FCCP and BAM15. As addition of the uncouplers reduce MTG values in flow cytometry.
I would like to ask if someone has observed the same using the
Nonyl Acridine Orange (Acridine Orange 10-Nonyl Bromide)?
Hi
Performing Westerns for VDAC or similar mt proteins followed by comparison of intensities may provide a better estimate. Even the copy number of mtDNA is not so accurate, let alone fluorescent staining. Uncouplers are unlikely to interact with fluorescent dyes, but CCCP and others do alter the fission /fusion ratio, resulting in uneven measurements.
By the way, if you think that CCCP / dye interaction is a problem, making FACS with a cell-free probes containing the same volume and concentration of uncoupler and dye may give an idea of a background .
Thought about TOM protein staining (nonviable, permeabilisation needed)?
I would suggest to run a citrate synthase assay instead of using fluorescent dyes. It is cheap, quantitative and convenient.
If you have antibodies against VDAC it's also a good way, as Alex suggested.
Regards, Christian
Hi)
You can try to add verapamil. May be it may help to preserve MTG. Article Dye-Independent Methods Reveal Elevated Mitochondrial Mass i...
By the way, I also experienced a MTG signal loss after treatment with FCCP in MEFs, but not in HUVECs .
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