Using confocal imaging, I'm investigating the effect of a particular compound on mitochondrial membrane potential in live cells. I've stained the cells with varying concentrations of the compound for a 10 minute time period and find a dose dependent decrease in TMRE fluorescence, indicating a decrease in mitochondrial membrane potential. At high concentrations of the compound, both TMRE and NucBlue are completely absent from the images. Since NucBlue is supposed to stain all cell nuclei (dead and live cell), I'm wondering why NucBlue disappears completely?
When using high concentrations of TMRE, it is possible to alter not only the mitochondrial membrane potential but also the overall cell membrane integrity, potentially affecting the cell's permeability to other compounds like NucBlue. It's also possible that high concentrations of TMRE could aggregate on cell membranes, potentially reducing membrane permeability or blocking NucBlue from entering. These are just some thoughts.
Vasilios Tsarouhas, Thank you for your response. The TMRE and NucBlue stain are apparent at t=0 in the control cells and in cells treated with lower concentrations of the compound that I am testing, but not in the cells treated with higher concentrations of the compound. Also, the treated cells have both TMRE and NucBlue staining at t=0 but lose both within 10 minutes, suggesting a loss of mitochondrial membrane potential and, possibly, cell death. I'm wondering if dead cells can lose NucBlue staining, but can't find any confirmation of this in the literature???
Thank you for your clarification. It seems that dye penetration isn't the issue I had previously thought it to be. It's hard to understand why dead cells do not stain with NucBlue. Could it be due to severe DNA damage or degradation, which affects (loose) the binding affinity of NucBlue to DNA? Or could it be caused by DNA fragmentation and leakage outside the cell? Have you tried to stain the "high dose" treated cells with Lamin to assess whether the nucleus maintains its normal morphology? or staining for histones ?
Vasilios Tsarouhas, I think that severe DNA damage, degradation, fragmentation and/or leakage are very plausible explanations for what I observe. I have not stained with lamin, but thank you for the suggestion!
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