Is staining time important?
If the answer is yes, I want to know if the damaged mitochondria by drugs are less-stained with the dye? Also, what are the detailed conditions such as dye density, incubation time, etc?
Hi,
it may depend on wether you are working on isolated mitochondria or with whole cells. By the way, internalization of most of the mito-staining dyes is driven by the transmembranar potential difference (delta psy), which is linked to the oxidative phosphorylation rate. As this function is one of the most (if not the most) important in mitochondria, any molecule impacting mitochondria functions (or membrane integrity) should impair the incorporation of such dyes, resulting in a weaker staining (or a shift in fluorescence emission wavelenght). Protocol may be slighlty different depending on your experimental system, but generally a µM concentration with incubation time around 30 min should promote a fair staining. For mitochondria activity (which one?) you can also try a ROS-sensitive dye as mitochondrial damaging often results in ROS generation due to the leak of electrons from electron transport chain. On isolated mitochondria you can measure oxygen consumption and respiratroy coupling with different substates.
in my experience mitotracker accumulates in cells permanently, therefore it is difficult to measure a reduction in staining, I would recommend either TMRM or JC-1
You can use dyes like CMXROS. They will be perfect for your needs. JC-1 also can be very useful.
MitoTracker Deep Red also responds to the mitochondrial membrane potential. Here is a paper showing that the mitochondrial uncoupler FCCP decreases mitochondrial retention of MitoTracker Deep Red.
The paper also shows that a switch from glycolysis to OXPHOS in cultured cells substantially increases mitochondrial retention of MitoTracker Deep Red. When testing for mitochondrial toxicity of drugs it's important to use cells which actually utilize OXPHOS. Most cultured cells grown in glucose-containing medium are predominantly glycolytic and therefore not a good model for testing mitochondrial toxicity.
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