Hi, you should not need to increase the glucose concentration from normal - in fact that could have unintended consequences. We have previously used glucose oxidase at 2mg/ml (in normal PSS) to generate extracellular peroxide in intact vascular smooth muscle preparations, but others have used as little as 10 microg/ml in cultured cells (again in normal medium). [Note these amounts are in grams rather than units] In neither case were anti-oxidants depleted, but these were relatively short term studies. See: Bell et al., Journal of Cell Biology, 177 (2007) 1029–1036; Pourmahram et al., Free Radical Biology & Medicine 45 (2008) 1468–1476).

The issue about timelines rather depends - glucose oxidase continuously generates peroxide (note that it does not generate superoxide, unlike some other pro-oxidant stimuli), but various factors including not just glucose concentration but also pH, temperature etc affect the rate of production. Acute oxidative (peroxide) stress is relatively simple - for chronic (several days?) I would replace the medium (including glucose oxidase) every so often. I would also use a method such as Amplex Red to measure peroxide in the medium at several time points throughout the study.

Hope this is helpful.

Thank you! It is helpful!

Look at the following protocol

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