I am using esophageal cancer cells and trying to detect ROS level with DCFDA and DHE dye using FACS, my results vary so just wanted to get some suggestion. Thanks.
DCF exhibits reactivity toward certain ROS but not others.
See: http://www.jbc.org/content/275/18/13175.full
Over time, the composition (or relative abundance) of ROS/RNS may evolve with passage. SOD for example is commonly overexpressed in cancer cells. So, over time, you may or may not be able to capture an ascending trend.
In addition, while DCF supposedly reacts with hydroxyl radical (see the above JBC), hydroxyl radical is known to react with solvents like DMSO. (See http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1153442/).
DCF is also chemically unstable in oxygenated environment over time.
Therefore, try to make sure the conditions are optimal and stable. Good luck.
Yes over time cancer cell lines could became highly resistance to apoptosis that is why you should keep you cell passages fresh and not grow them ( i mean for a single passage over 3 months) . See the spec of you cell line and grow them accordingly. Do not overgrow the cell line. Happy holidays!
Some cancer cell lines their properties markedly. Some may undergo diferentiation proces which may result in various ROS level during the treatment.
Good luck.
P.M:
Sorry, the reference on DCF selectivity should be this JBC, not the previously listed one.
http://www.jbc.org/content/278/5/3170.full
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