Looking for Superoxide / Peroxide Detection Methods - (Oct/18/2005 )
I need to quantify the degree of "reactive oxygen species" supression induced by an anti-cancer compound in our lab. In particular, I am interested in monitoring superoxide levels, but peroxide concentrations may also be important, since the two are related through dismutation.
So far, both with live cells and lysates, I have found the traditional methods (cytochrome C, NBT, phenol red oxidation, etc.) to be rather lacking in specificity. The problem is that all of these methods respond to other species in the milieu, such that the signal I observe is more due to general cell metabolic health than it is to ROS concentration.
Does anybody know of a more selective method for determining either superoxide or peroxide?
I know of a pretty good way to detect peroxide in lysates but not in cells. We use a kit called Amplex Red Acetylcholine detection kit. The final step of the reaction used in this colormetric assay is that of Horseradish peroxidase on H2O2 reacting with their compound resorufin turning from a clear to purple-ish. compound. You can get this kit from Invitrogen. In your case you would not need to add the other enzymes (choline oxidase and acetylcholinesterase in the mix. I don't know however if you can get just HRP and resorufin individually. Activity is based on the fluorescence (excitaton ~545 and emmission ~590). Hope this can be of help.
I shall order some resorufin (already have lots of HRP).
Still looking for a better superoxide method if anyone knows one.
What we do in our lab to monitor the levels of ROS is to measure the extent of the damage they've induced. In our model, astrocytes and motor neurons, we perform IHQ against nitrotyrosine, a footprint for peroxynitrite mediated damage.