What are the dyes which specifically detect Hydroxyl radicals and hydroxyl ions in cultured cells.
Likewise, what are the specific inhibitors of superoxide anion formation and H2O2 formation.
There are no useful methods for this, in spite of many claims. The reason is the high reactivity of these radicals which will react in preference with the major cell components - proteins, nucleic acids, carbohydrates, metabolites, etc. Von Sonntag (Methods Enzymology, vol233(1994) 47-56) has shown it is impossible to introduce sufficient concentrations of an indicator into cells to react preferentially with hydroxyl radicals.
Thanks for the information Jan. Could you suggest any method for the detection of cytosolic and mitochondrial superoxide anions in cells.
I think the best way is to measure hydrogen peroxide - there are several good methods see Murphy Biochem J. 417 (2009) 1-13 and follow the references.
Jan is correct. However, there are some EPR technologies that might be useful. However, these require specialist instruments and an expert to run your samples. The analysis of H2O2 on the other hand is much more stable and easier to measure. Stay away from DCF since this is not very good. MitOSOX for mitochodrial superoxide is particularly easy to use if you have a flow cytometer, or UltraAmplex Red for H2o2 using fluorescence plate reader
Hydroxyl free radicals, no doubt, are ESR active (presence of one unpaired electron), but are very unstable and may recombine to giveESR inactive H2O2 and the ESR activity of hydroxyl free radicals may vanish at all or decrease.
This limitation can be overcome as follows:
When a spin trap like TEMPO (2, 2, 6, 6-Tetramethylpiperidinyloxy free radical) obtained from TEMPO-H is added, it forms a relatively stable free radical with the unstable hydroxyl free radical while keeping its spin intact (still has unpaired electron) So the recombination of OH free radicals to form H2O2 is prohibited.
TEMPO-H+OH (Free radical)-(-H) --TEMPO- OH (Stable free radical).
2OH (free radicals) ------ H2O2 [prohibited].
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