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Talk:Measuring hydrogen peroxide

From Bioblast

MiPNet discussion forum: hydrogen peroxide production in intact muscle fibers (2013-10-25)

Marcus Olivera

Iยดve been working on intact insect flight muscle to evaluate respiratory capacities but Iยดm experiencing many problems in detecting changes in rates of Amplex red fluorescence on physically permeabilized flight muscle. Does anyone know a suitable method to detect hydrogen peroxide generation on intact muscle fibers (not isolated mitochondria)?


Nicolas Pichaud

We are presently doing a test with a marker called CellRox from Molecular Probes but you need a confocal microscope. It is working pretty well so far, we just need to do a bit more of optimization... I'll send you the protocol when it is ready.


Daniel Munro

Permeabilized fibres still contain most of their antioxidants. I wonder to what extent they compete with Amplex Red for the H2O2. The results will certainly not represent actual rate of mitochondrial ROS production but rather a combination of ROS production and the strength of the antioxidant defences.


Chris Hedges

To follow on from Daniel above, have you used positive controls to ensure you can detect produced superoxide? Such as adding xanthine+xanthine oxidase - this will tell you whether it is the assay or the model which is not allowing you to detect fluorescence. But it could well be that flight muscle antioxidant levels are outmatching the rate of superoxide production.