Dear, Evryanna. 

In a similar study, we introduced hydrogen peroxide to a final concentration of 400 uM to the culture of peripheral blood lymphocytes. We did not change the medium for the next 48 h - until harvesting the culture, as we studied changes in protein expression. 

Please note, that 400 uM is not a way too high concentration, so it does not significantly decrease cell viability. However, the extent and the type of oxidative stress induced must be determined for all experiment series. From my experience, it does induce oxidative stress, but the stress is weak and is of antioxidant-depleting nature. 

Hi I've done this sort of thing in 3D matrices (so not PBMC's) and we used a range of H2O2 concentrations 0, 200 mM, 1 mM or 2 mM H2O2

Hydrogen peroxide is not very stable and decomposes quite rapidly I believe so you may want to either renew it in the media periodically or use a more stable "stressor" (such as Menadione, a quick "Google" shows there are several articles using this compound and PBMCs you might want to look into).

Hope this helps,

Gary

First check the viability of PBMC in different doses of H2O2 for 24 hrs and then take the  best of it and do a time point of it say, 4,8,16,24 hrs and then you can plan your experiments further on the basis of ROS generation

Dear Evryanna,

We have just finished an experiment that may be of interest for you. Almost as in our previous study, we treated cells (HeLa this time) with H2O2 (final concentration of 800 uM; single treatment) and harvested them after 24 h for an RT-qPCR analysis. Control cells were treated with H2O and followed the same procedure in the rest. According to preliminary data, this treatment efficiently causes oxidative shift and the adaptive response in the cells. However, the cell devision rate was significantly lowered in the treated cells.

Hi, I think an option available to you would be to use GOX (glucose oxidase).  You can use this enzyme to in variable concentrations to create biologicial relevant concentrations of H2O2.  As stated above, you will need to titrate out the amount of H2O2 since low levels canl promote proliferation and once this threshold is exceeds your cells will begin to die.

Dear, Evryanna. 

Herewith, i have attached the article and i hope it will be useful for you