I am currently measuring ROS in T24 bladder cancer cell after drug treatment. I have already measured ROS generation by microscopy using cmDCFDA. It is confirmed that my drug induces ROS as NAC treatment reduces DCFDA fluorescence but, I am unable to monitor the same by FACS. Precautions to be taken while FACS are also welcomed.
DCFDA should fluoresce in FL1, leaving the other fluorochromes for cell markers. I would stay away from using PE as DCF can get messy. Leave that channel open. As well, monitor a couple of diff. concentrations of NAC, as too much will give a prooxidant response noted by increased DCF/ROS levels.
Stain cells with markers first, THEN DCF. If it is a cell line, you probably wont need a marker, just the DCFDA then.
Use a 5uM concentration of the DCFDA and incubate 15 min in a PBS/Facs buffer at 37deg. in the dark, wash then run on the flow within the next 30 min. Measure by MFI(mean fluorescence intensity) fold changes. Please post any other questions, issues you may have and good luck!
Hello Mohammad.
Some years ago we published a comprehensive series of method to detect oxidative stress and mitochondrial function using flow cytometry.
I would strongly recommend to use a multiparameter method. In your cells there id also an high level of autofluorescence which should be taken into account.
http://www.sciencedirect.com/science/article/pii/S0014482798943439
http://www.nature.com/nprot/journal/v2/n12/full/nprot.2007.397.html
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