I'm trying to measure the ROS production from RAW 264.7 cells by using DCFH staining and analyst by flow cytrometry. The problem is the level of ROS production in control cells was high. I'm not sure what am I doing wrong. Can anyone help me solve this problem?
Many years ago (four decades) I used a DCFH assay to look at the levels of H2O2 within trypanosomes, other workers using this assay and extrapolating to obtain an internal H2O2 level, had calculated an extremely high internal level of 70 micromolar. This value was clearly wrong as 70 micromolar external H2O2 was rapidly lethal (the true intercellular value is in the low nmolar range). This assay (DCFH) was developed to determine the levels of H2O2 in antarctic snow as a measure of its exposure to sunlight (O2 oxidizes water to give H2O2 when exposed to UV radiation). Using this assay I could detect H2O2 in the labs distilled water carboy which sat in a sunny location for much of the day. I therefore abandoned this assay due to its unusable high sensitivity, which would require dilution of samples with truly H2O2 free water/buffers, in favor of a less sensitive assay. I would recommend using trapping assays for various ROS species, and measuring the products generated on interaction with biomolecules, to determine ROS exposure.
Thesis Nitrofurazone and Hydrogen Peroxide Metabolism in Trypanosoma brucei.
Article Hydrogen peroxide metabolism in Trypanosoma brucei
I hope this helps, and good luck in your research.
Phil
DCF is very sensitive. I will suggest you optimize the concentration. Most papers report usage in the 5 to 10 micromolar range but you could reduce the concentration further. Ensure your samples are kept away from light and analyze promptly after staining.
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