If I want to induce oxidative stress by adding H2O2, what percentage of it should be added and at what temp? if I am doing imaging of cells before adding the H2O2, continue adding H2O2 directly while doing live imaging or so?
please explain.
I would attempt a dose curve, as different cell lines have various thresholds of sensitivity to hydrogen peroxide. A very typical dose of inducing stress is in the range of 1uM-100uM. This usually induces stress without killing the cells dramatically. Some cells can handle ranges all the way from 500-1000uM as well, but most you will see death over time (especially if you are imaging them).
I would start at 1uM and go up from there. You can do this at room temp, but it really depends on the assay you are using. What assay would that be?
Hi, Megna, plesae, consider that not only cell type, but also stage have diiferent threshoold for H2O2 senitivity. However, the concetration is alos largely dependent form the aim of your experuemnts. If you aim is kinetics of cell death/damage, you can use hiher dose, but in the case if your aim is cells re-programming (adaptation to stress conditions), you should to start with low dose and investigate specific pathways induced by H2O2. It is will benefical if you add H2O2 in condition medium, but do not add H2O2 with new medium. Good luck!
Thanks Taras sir,
Condition medium means only the plain medium or with FBS?
Megha Ji
Actually I have not used the neuroblastoma cells for H2O2 in duced oxidative stress. However I had used the PC12 cells for the same purpose. For PC 12 cells the method is as follows:
For H2O2 treatment, PC12 cells were washed with FBS free DMEM. Dilution of H2O2 was made from a 30% stock solution into DMEM just prior to each experiment and 200 μM solution added to the cells. The culture plates were incubated for an additional 24 h. Samples of different concentrations were treated 2 h before and during H2O2 exposure. Control group (without H2O2) was incubated under
the same conditions.
if you are interested in this method you can just go through the following article and all the best.
http://www.mdpi.com/1420-3049/18/3/3529
Thanks! Fresh medium changes cell metabolism significantly, required formation of new gradients, changes stress-reletd enzyme activities. Such changes required at least 2-3 hours for adaptation. Briefy, fresh medium is some kind of stress.
So, it is better to avoid such changes.
My best wishes!
Taras Pasternak Thank you SO much for your reply! Sorry, I still have two questions to bother you.
#1 If my the exposure time of my interested exposure is 24 h. Since the "H2O2 in medium is generally cleared within a few hours", should I keep the positive control group exposed to H2O2 for 24 h(consistent exposure time)? If not, how long will you suggest?
#2 For adding the H2O2 in condition medium, do you mean to add the certain volume of 30% H2O2 stock solution directly into the cultured medium? Will the local high concentration of H2O2 be bad for the cells? If you don't mind, could you please share me with your protocol?
Many thanks,
Best wishes!
Thanks! The H2O2 stability in the medium were dependent form many factors (pH, ions, natural H2O2 realizing to the culture medium by cells) etc. It is better to try to measure H2O2 concejtration after several time point (2-6-12 hours, for exmaple) and thereafter decide about addition of extra H2O2.
If you used liquid medium, H2O2 can be quickly distributed in the medium after addition, so, it is not a big probelm. But you can dluted stock to 5% before addition.
You can also used tBHP which is more staböle as H2O2. Plesae, have a look here and similar papers.
My best wishes!
Article Tertiary butyl hydroperoxide induced oxidative damage in mic...
You can try DCHBS/AAP method what give you quantitative data in real time