I stimulated the cells with CCCP (10uM) as well as CCCP + Chloroquine (50uM), as well as having an unstimulated sample. The Mitotracker DR signal massively increased when I added CCCP.
I know the signal is meant to decrease with CCCP, as the mitochondria are removed from the cell via mitophagy, and the addition of chloroquine was meant to prevent their removal.
Any reason why it might have increased?
I used a DAPI live dead solution to only measure the live cells
Just some thoughts: Deep Red is believed to be a membrane potential sensitive dye. Perhaps the increase in signal could be a (i) transient stage before removal, (ii) the result of selective mitochondrial removal, leaving "good", highly polarized mitochondria intact or (iii) due to a timing issue. If possible check earlier and later timepoints for signal intensities.
You may also need to tweak the concentrations in your setup to see what you want to study and make sure the dye is not expired.
How long are you using of CCCP? In my experience, I got the same result. For shorter time like 30 minutes, MMP is reducing but after a while like 4 hours, It starts to increase.
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