I have found several research papers that used DCFH2-DA to evaluate oxidative stress through ROS formation. However, different concentrations of DCFH2-DA are indicated, ranging from 1 uM to 100 uM. Thus, I was wondering if there may be a possible interference when using high concentrations of DCFH2-DA, e.g. high concentrations may somehow block the uptake of the test compound.
Thanks in advance, and I would appreciate any comments.
After being taken by the cells, DCFDA itself is deacetylated by cellular esterases to a non-fluorescent compound, which is later oxidized by ROS into dichlorofluorescein (DCF), a highly fluorescent compound. DCF can be detected by fluorescence spectroscopy with maximum excitation and emission spectra of 495 nm and 529 nm respectively. Its uptake according to literature is through diffusion and if uptake of your compound is through a transporter or some other mechanism, then i's unlikely that DCFDA affects the uptake of your test compound.
DCFDA is not a specific ROS probe. It can be converted by various cellular components that are not strictly related to oxidative stress. Please refer to the attached papers for more details. For example, Amplex Red or MitoSOX are better ROS probes.
Regards, Christian
Article What does the commonly used DCF test for oxidative stress really show?
Article Measurement of Reactive Oxygen Species, Reactive Nitrogen Sp...
Thanks for your comments.
Christian Q. Scheckhuber, thank you for the references. They are very helpful!
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