I tried to measure palmitate-induced ROS with DFCDA in H9C2 (10uM) but even though I tried several protocols (with PBS, washing prior labelling, washing after labelling, using 2X DCFDA directly in the wells with treatment in order to not remove the treatments...) I never see differences between control and treated cells. I know for other researchers that palmitate produce ROS and in the literature DCFDA is widely used but I can't find the appropriated conditions.
1. If you might know a solution I would appreciate you share it
2. I want to use mitosox because it's more specific of mitochondrial ROS and also it seams to be more stable and efficient. Any suggestion?
First if the 10uM is that of the dcfda, it might be high imo. try reducing it to 2.5
second, adapt the procedure according to your instrument used for measurement (facs or a plate reader).
for a plate reader, you will be growing your cells in a 96 well plate.
1- wash cells twice with warm PBS
2- add the dcfda (2.5uM final concentration) to PBS containing 0.5mM glucose
3- add the mixture to your cells, 200uL / well
4- place your plate in the plate reader and measure for 45 min, 1 read out each min.
Good luck!
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