Dear Reinold Goetz: We have published few  papers in which monolayer Huh7 cells  were treated with H2O2  to induce  ROS and stress signaling cascade and here is the reference for details on the methodology.

Qadri I, Iwahashi M, Kullak-Ublick GA, Simon FR. Mol Pharmacol. 2006 Aug;70(2):627-36. Epub 2006 May 2.epatocyte nuclear factor (HNF) 1 and HNF4 mediate hepatic multidrug resistance protein 2 up-regulation during hepatitis C virus gene expression.

IIshtiaq QADRI, Mieko IWAHASHI, Sonia FLORES, Jerome SCHAACK

Journal: Biochemical Journal - BIOCHEM J , vol. 378, no. 3, 2004 Induced oxidative stress and activated expression of manganese superoxide dismutase during hepatitis C virus replication: role of JNK, p38 MAPK and AP1

Hi, it depends on which ROS you want to measure ?  Sure if you measure the H2O2, you should'nt treat your cells with it! If you want to image ROS production you can use the glucose/glucose oxidase pair to induce oxidative stress or any drug/chemical known to induce ROS production (ETC inhibitor for example). Indeed ROS production can be estimated by fluorescent dyes or SOD activities, for example.

Dear Reinhold,  Hydrogen peroxide will slowly oxidise reduced methylene blue in solution.  ROS will accelerate the reaction.  You will have to experiment with conditions to find the optimal levels.  

Recommendations for H2O2 as a positive control. H2O2 is destroyed by cell culture components (possibly pyruvate), addition in PBS? Then H2O2 will be partly destroy inside the cells if you add it long time before measurement, by normal cell defense (addition of 1 µM, one hour, you can still detect what is left). Then, H2O2 addition will change the balance of ROS inside cells, so they will be an increase of other ROS, for example it precursor superoxide anion (so you detect increase of H2O2 but also other ROS, depending on the technique). Hope it help!

Hi Reinold, I agree with all the comments before me. If you want to measure H2O2 (end product of ROS) only, I would suggest a spike of H2O2 (different concentrations to construct a dose response curve) for a short time, since H2O2 can penetrate membrane freely and will be destroyed quickly (like hours) either by intracellular antioxidant system or reaction with protein etc. If you like to measure chronic effect of  ROS, the glucose/glucose oxidase pair will be my choice. We usually use glucose oxidase (1-3 mU/mL) in high glucose DMEM medium as a model to examine protein carbonylation. Hope this will help. Jiang

 Re: measurement of ROS generation, you could consider a ratiometric dye such as ro-GFP (reference, Waypa et al - PMID: 21670147) as opposed to a single wavelength dye such as DCF. Another idea would be, as mentioned earlier, to consider using an endogenous activator of intracellular ROS generation. This is cell type specific, but for instance, you could consider an NADPH oxidase agonist for certain cell types. In our hands, H2O2 concentrations are stable for a couple of hours in certain solutions but only if the solution is serum and trace metal free such as Krebs (reference: Suresh, et al. PMID: 26453519)

Another consideration is that only small proportion of added H2O2 (up to around 15%) will be biologically available intracellulary. This is probably due to the reasons highlighted by Ann-Cecile above, and possibly limited membrane diffusion. Endogenous catalase, glutathione peroxidase etc will also destroy intracellular H2O2.

Whether ROS generation is your aim by any agents or ROS generation by any other agents will be OK with you? I have used TNF-alpha and bacterial LPS. They are quite effective in ROS generation.

Hi also using xanthine oxidase system as spike or positive control in your media without/with cells is possible. It depends if you want to measure H2O2 or other radical forms of oxygen.

Whether ROS generation is your aim by H2O2 only or ROS generation by any other agents will be OK with you? I have used TNF-alpha and bacterial LPS. They are quite effective in ROS generation.

I agree with Dr. Vidal and Dr. Mukherjee. It's better to induce endogenous ROS, rather than using an exogenous ROS to augment them, because species like H2O2 have broad and nonspecific effects if applied globally. Even GSH will be reacted upon. It's hard to say what's being mimicked. 

Menadionewhich induces intracellular ROS is a lot more accurate than hydrogen peroxide.

Thank you all for your input! I needed a positive control to make sure there wasn't anything wrong with my experimental technique (attempting to induce ROS using LPS stimulation).

Well LPS will induce also iNOS, so maybe I would also use L-NAME to inhibit NO production and subsequently peroxinitrite formation (after reaction with superoxide) prior any ROS measurements. Superoxide can be scavenged by high levels of NO.

Out of the two agents (TNF/ LPS) I have mentioned I prefer to use TNF-alpha. In my case 10 ng/ml of TNF-alpha (final concentration in the medium M199) for upto 4 hr treatment for HUVEC generate maximum amount of ROS

What is the question you are asking, the hypothesis that you are testing? Are you interested in the physiology or pathophysiology, the molecular biology of chronic or acute oxidative stress? I assume you using cells in culture, if so what cells are you using and what oxygen tension do you subject your cells to?

I suggest that you read my critiques of the use of cell cultures in 19% O2 and H2O2 to study the physiology, pathophysiology of oxidative stress and their molecular biology in the discussion section in our 2012 publication (1) (not just the abstract).  Also our first follow up publication (2).  There are a plethora of studies using such approaches.  I question their value, recognize their limitations and move on.  !9% oxygen is not physiological since cells in tissues are under at most 5% oxygen and cells grown under  19% O2 are adapted to chronic oxidative stress.

If you wish we could have a discussion of these issues may be via Skype once you know where I am coming from.

1.         Weisz J, Shearer DA, Murata E, Patrick SD, Bing H, Berg A, et al. Identification of Mammary Epithelial Cells Subject to Chronic Oxidative Stress in Mammary Epithelium of Young Women and Teenagers Living in USA: Implication for Breast Carcinogenesis Cancer Biol Ther. 2012;13(2):101-13.

2.         Patel, II, Shearer DA, Fogarty SW, Fullwood NJ, Quaroni L, Martin FL, et al. Infrared microspectroscopy identifies biomolecular changes associated with chronic oxidative stress in mammary epithelium and stroma of breast tissues from healthy young women: implications for latent stages of breast carcinogenesis. Cancer Biol Ther. 2014;15(2):225-35. doi: 10.4161/cbt.26748. PubMed PMID: 24107651; PubMed Central PMCID: PMC3928139.

You can incubate your cells with 100 µM menadione for one hour or 50 µM nefazodone for 24 hours, as positive control for intracellular ROS reaction.

However, H2O2 can also be used, though it does not work well for CellROX dyes.

Hi Reinold,

people often use H2O2 as a "positive control" but I would rather not. If you give a substance in your well that you want to detect with your probe, of course you will get a signal. But you want to know how and if your cells are producing ROS. It would be the same as if you would put IL-6 in your IL-6 ELISA and of course you will get a "IL-6-Signal".

The suggestions that were given before offer good hints already. It highly depends on your experimental setting. Not all cells produce ROS in the same amount, in all compartments with every stimulus. What induces ROS in one cell type might not induce or even decrease ROS production in another or perhaps with other kinetics. If you know a stimulus that works in your other experimental read-outs and this stimulus does not induce ROS production with the same concentration, then there is simply no ROS production that can be detected.

The only reason one should add H2O2 to cells is, when the oxidative stress response is the scientific topic. In all types of ROS measurement settings this does not make sense. The commonly used probes (Isoluminol, Luminol, DCF derivates) work and will detect ROS if the cell produces enough.

I hope that helped you. For further technical details for ROS measurements, please have a look at our paper. More than 70 % of it is more or less ROS measurement techniques in different compartments, with different stimuli or scavengers (with macrophages though).

Mitochondrial reactive oxygen species enable proinflammatory signaling through disulfide linkage of NEMO, Science Signaling (2019)

All the best,

Marc

In context of this topic, I would like to bring to your attention a recent review of our group, "Functions of ROS in macrophages and antimicrobial immunity".

In this review, we give an introduction to ROS and their sources in macrophages, summarize the versatile roles of ROS in direct and indirect antimicrobial immune defense and provide an overview of commonly used ROS probes, ROS source inhibitors and ROS scavengers (also the difference between ROS scavengers and antioxidants, which are not synonymous, is explained).

If you like, please have a look at: Functions of ROS in Macrophages and Antimicrobial Immunity February 2021, Antioxidants 10(2):313 DOI: https://doi.org/10.3390/antiox10020313

Please also have look at our Perspective article, if you like:

Article Reactive Oxygen Species: Not Omnipresent but Important in Ma...