I am treating my cells with a drug that generates about 20-40 uM H2O2. I see a profound activation of gamma-H2AX but no/very low levels of corresponding DNA damage in comet assay.
I am trying to find out if the induction of gamma-H2AX is somehow related to the generation of H2O2 in my drug treated cells.
It depends on your system. H2O2 has to penetrate into the cells through the membrane and then goes to nuclei. I would suggest trying a series of concentrations and monitor the markers you use. The following discussion may also help:
https://www.researchgate.net/post/How_to_estimate_the_induction_of_ROS_in_cell_culture
Thank you Shin Murakami for your reply. I have added a bit more information/ data to my question to clarify what I am looking at.
The following paper may be helpful:
Article Dynamic Monitoring of Oxidative DNA Double Strand Break and ...
Article Fully automated interpretation of ionizing radiation-induced...
Phosphorylation of histone, forming gamma-H2AX, is an early response to double-stranded break (DSB). H2O2 can cause oxidative damage on DNA but it unlikely causes DSB directly. One of the papers talks about H2O2 and may give you a hint.
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