Hi all,
I am trying to study the effect of oxidative stress on my reporter gene activity, in hek293 and hela cells. I searched a lot to see if anyone did something similar, but couldn't find any. However, people studied oxidative stress using different concentrations of hydrogen peroxide (0.05mM, 0.1mM, 0.2mM, 0.5mM, 1mM) with 24hr incubations in other cell lines. Cell death/apoptosis was reported with 1mM treatments.
I need advice for, if I treat with 0.5mM hydrogen peroxide for 2hr, will this be okay (for starters)? Also, did anyone use any other small molecule to induce oxidative stress in mammalian cultures (concentration, treatment time etc)? TIA.
P.S: I am NOT looking for induction of apoptosis.
Hello Shruthi
I have studied the cell death and survival by oxidative stress with hydrogen peroxide in human monocyte/macrophe. I advice you try with 3 concentration of hydrogen peroxide (0.1, 0.5 and 1mM) and analyze the cytomorphology at 2 , 4 and 6 hours. Then, you can choose the condition where observe the apoptosis signal at 6 hours. After, analyze to RNA level of BAX/BCL2 expression at 2, 4 and 6 hours, as well as anlyze CASP3 protein or other that you consider too. In this way you can analysis the level of oxidative stress and consider which is the best for you study.
Actually, cell culturing conditions themselves give oxidative stress because they are cultured in a much more oxygen-rich atmosphere than their native environment in the human body. Instead of using hydrogen peroxide as additive, you can try to work the other way and culture the cells in an oxygen-poor atmosphere.