Hi everybody!
I got some strange results when I tested mitoSOX RED on Bone marrow-derived Macrophages.
According to the publications, bone marrow-derived macrophages(BMDMs) generally show increased mitochondrial ROS production after LPS treatment.
However, when I use mitosox as a mitochondrial ROS probe to run a test on BMDM, I have always found that LPS treatment results in a significant decrease in the fluorescence intensity of Mitosox Red. I have tested LPS for a variety of time courses and at different concentrations. All LPS-treated groups show a reduction in fluorescence intensity. This is the exact opposite of what has been reported in the literature. Here are some of the reagents I used to culture BMDM:
RPMI-1640 Glutamax + 10% FBS + sodium pyruvate additive + HEPES + beta mercaptoethanol(55uM at final concentration) +M-CSF (20ng/mL).
I am currently considering replacing the medium with one that does not contain beta-mercaptoethanol because it may impact ROS.
If you have any ideas about this experiment, please give me some advice.
Just keep very simple media of RPMI+serum, stain at 37C for 30 mins. Should resolve things.
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