Hello. We also used H2O2 because it is so cheap, or? What concentration do you add? generally it should be possible to work with 1 % H2O2.

The toxicity is the activating factor as ROS are toxic, so I guess this can not be prevented. However I would try to figure out the smallest amount of H2O2 suitable for positive control.

Franz-Josef Schmitt I'm using 100 uM of H2O2 for 24 hours in RPMI-1640.

That is a reasonable value - but then the prize should be neglectable. We used just standard H2O2 and diluted it:

https://www.carlroth.com/de/de/von-a-bis-z/wasserstoffperoxid-30-%25/p/cp26.1

Dear researchers, it looks to me that you do not understand what oxidative stress is. H2O2 is a byproduct, usually in a very small amount, which accompanies mitochondrial production of superoxide radical. Yes, there are reactions, mostly in the liver, that produce relatively high levels of hydrogen peroxide. But pouring H2O2 into culture media is an old habit, but a rather unproductive method to study oxidative stress. Do you think that the hydroperoxide you have added lives long enough to do any harm to cells? In addition, your culture media contain PenStrep, which kills mitochondria. Than what do you study? I have observed that just in a medium without any biological product the rate of hydroperoxide increased and was depended on the atmospheric content of ozone. To study the effects of H2O2 is the waist of time.

Dear Dr. Alexander V Panov , as I understood H2O2 was not used to mimic oxidative stress where I fully agree but used as a positive control for the functionality of the H2DCFDA Assay that i activated by H2O2.

OK, his is an indicator for the presence of H2O2, but itis a qualitative indicator. Many radicals serve as a cellular signals for some metabolic events in the body. But what sense the indicator has when use a bucket of H2O2 instead of small amount? I have worked with many cells, when studying prostate cancer, and became highly disappointed with culturing cells. Too many simplifications and simply unacceptable errors and too far from the complex process of oxidative stress.

HI Sandeep, the radicals produced using H2O2 depend on the availability of Fe for the fenton reaction. If you want the cell's mitochondria to produce ROS you can add Menadion (Vitamin K). at a concentration of 50uM it will induce Apoptosis in the long run but also increase ROS. Your 24h time is probably a challenge if you want to add the positive control for that amount of time you might end up with a lot of dead cells. It is usually better to measure ROS production kinetics, that way you might be successful in detecting differences after a few h. see also https://www.jbc.org/content/278/15/12645.long

Best

Heiko

Prof Alexander V Panov

What chemical do you sugest to induce oxidative stress as positive control in cell culture?

As a matter of fact I do not study cell for 23 years now. this is mostly waste of time and your life. Particularly, if you grow up cells in the presence of PenStrep. My colleagues, who use cells, told me that after they stopped using antibiotics, as I have recommended, they started to obtain completely different results. Yes, it is more complicated, but these are results that you can comprehend.

Oxidative stress is a very complicated issue. Every tissue, every organ and every conditions affect the results. In addition, what radical you keep you mind when you thing about oxidative stress? It depends on the type of radical.

Under ordinary conditions, neither oxygen radial has much importance for the cell's functions. Except perhydroxyl radical, the protonated form of superoxide. It is HO2 that causes aging. H2O2 has much effects in the cuvettes you use to study cells and in vitro. How much is hydrogen peroxide in you medium? A lot!

Particularly, if you use Milli-Q water. Read my book "Practical Mitochondriology". You can see it on my page in Research Gate or buy for 5 dollars as a book on Amazon (Kindle).

Dear Rarto Widodo, you questions reveals that you have a vague idea what actually is oxidative stress. Things have changed vay muc recenltly. I have published severaal papers, which you can find on my pare on RG.

Good luck. Read more.

AVP

Thank you very much, Prof. A.V. Panov

Yes, sure that oxidative stress is a very complicated issue. Many conditions affect the results.

If all conditions are strictly maintained at the same status except for the treatment for example h2o2 concentration, that the result of the treatment scientifically can be compared and discussed.

If that so as you explained anyway.... What do you suggest to study oxidative stress? What the most proper method do you consider related to oxidative stress study?

I would not advise using the DCFDA assay for measurement of oxidative stress, it is a very non specific measurement and depends on a huge number of factors from esterase activity, transition metal availability and mobilisation. There are spin traps for hydrogen peroxide available on the market for use with EPR.

If you absolutely have no choice but to use DCFDA, mitochondrial uncouplers such as CCCP are often used as a positive control.

Prof Panov makes a good point about the issue with adding h202 to culture medium. A significant portion will react with things such as transition metal ions through Fenton reactions.

H2O2 is good +ve control...

Sourbh Garg Sandeep Kumar

Hello, I have started ROS experiments and am facing some trouble. At what conc. of H2O2 you used and how much cell death did you find?

Hello, I sometimes use glucose oxidase as a source of H2O2 instead of adding H2O2 directly; it can generate H2O2 for a longer time than direct addition of H2O2. For detection by DCFDA, it can be detected by the usual method after removing the culture medium. However, when looking at the movement of signal transduction in a short time, H2O2 was added directly.