Cells have natural defences against hydrogen peroxide, namely, catalases peroxidases and glutathione being predominant . So low H2O2 < 50 uM may have zero effects on oxidative stress as the natural defences will destroy the excess H2O2 beforecan exerts any oxidative effect. This can be demonstrated by using catalase inhibitors which enhance H2O2 toxicity at low concentrations.

In contrast high H2O2 concentrations > 1 mM can destroy cells altogether, so that a dose response will be lost with high H2O2 concentrations also.

Additionally as hydrogen peroxide has a short half life when in contact with metal ions its nominal concentration may be far in excess of the real concentration within the cells.

The reasons:

hydrogen peroxide has very short life

self defense of cellular metabolism to hydrogen peroxide

concentration of hydrogen peroxide (too low or too high)

incubation time

And: So you may need to vary the range of hydrogen peroxide concentrations as well as incubation time for better results.

And also you have to use serum free and protein free medium to induce stress.

Richard J Naftalin Sir, Thank you so much for your reply. I have incubated cells with 0.5, 1, 2, 3, 4, 5, 10, 20, 30, 40, 50, 100 and 200mM of H2O2 for 2h. For 0.5 and 1mM cell viability is more than 50%. From 2 to 20mM the cell viability is in increasing order!!!. As you said high H2O2 concentrations > 1 mM may destroyed cells altogether, so that I'm not getting dose dependent response.

Sasya Madhurantakam

Thank you so much for your reply akka. I have used serum free medium only. I have checked the viability after 1, 2, 4 and 24h of H2O2 incubation. The cell viability trend remains the same.

I'll try with concentrations between 50um to 1mM.

Thank you for the above questions and answers. I was also searching same since long time..

I am extremely sorry rather than giving answer, i have similar question here.

I used 400uM of H2O2 for inducing senescence in gingival epithelial cells. In 35mm, 60mm and 100mm of culture dish, only 10% of cells dies after 24 hours of hydrogen peroxide treatment, but 50-60% cells dies at 96 well plate for viability assay. What might be the reason? I have repeated it 2-3 times, still result is same.

Please suggest me.

Interesting question... H2O2 is one of the longest lived ROS products but will eventually break down to H2O and water.... so the access to cell culture medium and air are important factors in its rate of decay and also exogenous antioxidant enzymes... peroxidases catalase SOD and availability of metal ions.

What is the ratio of cell number: medium volume in you 96 well plate versus the other plate sizes?. what is the medium surface area: volume ratio in the various conditions.? It would be useful for you to determine the H2O2 concentrations in your various solutions after 12 and 24 hours... I suspect that this will supply the answer to your question....

Harald Kühnel

1 uM ferric ammonium citrate... iron speeds up the Fenton reaction by many orders of magnitude.... see Miller DM , Buettner, GR, Aust, SD Transition metals as catalysts of "autoxidation" reactions. Free Radical Biology & Medicine, Vol. 8, pp. 95-108, 1990