Extracellular H2O2 is relatively easy to measure, as I would recommend the Amplex Red assay. It is highly specific and relatively sensitive for H2O2, and should work fine on in vitro stimulation assays.

Intracellular H2O2 is very tricky. People will recommend you to use the fluorescent dye DCFH, but take it from a free radical biologist that this is garbage and not specific. Your best bet are the H2O2 sensitive fluorescent proteins like HyPer or the newly constructed boronate probes by Chris Chang's groups (not commercially available). Both of these come with specific caveats on the methodology, and are not totally user friendly as the technique needs sufficient optimization.

Is there a reason you need to look at intracellular versus extracellular specifically? I would recommend performing the Amplex Red for extracellular, and maybe a different more specific dye for other ROS intracellular (e.g. DHE or mitoSOX for superoxide). Very rarely is H2O2 produced as a direct product of stimulation, and is usally a downstream intermediate or signalling molecule after one-electron processes take place (i.e. superoxide generation).

You can also look for evidence that H2O2 was present such as lipid peroxides or protein carbonyls, but once again these are not perfect and not specific for H2O2.

You have a few angles to go for, but just really look into the specificity of the assay before you use it. Too many people use the dyes inappropriately and call oxidation whatever they want to see.... obviously without the proper controls (e.g. antioxidant supplementation or over-expression) this is not correct.

Good luck,

~Adam J. Case

Extracellular H2O2 is relatively easy to measure, as I would recommend the Amplex Red assay. It is highly specific and relatively sensitive for H2O2, and should work fine on in vitro stimulation assays.

Intracellular H2O2 is very tricky. People will recommend you to use the fluorescent dye DCFH, but take it from a free radical biologist that this is garbage and not specific. Your best bet are the H2O2 sensitive fluorescent proteins like HyPer or the newly constructed boronate probes by Chris Chang's groups (not commercially available). Both of these come with specific caveats on the methodology, and are not totally user friendly as the technique needs sufficient optimization.

Is there a reason you need to look at intracellular versus extracellular specifically? I would recommend performing the Amplex Red for extracellular, and maybe a different more specific dye for other ROS intracellular (e.g. DHE or mitoSOX for superoxide). Very rarely is H2O2 produced as a direct product of stimulation, and is usally a downstream intermediate or signalling molecule after one-electron processes take place (i.e. superoxide generation).

You can also look for evidence that H2O2 was present such as lipid peroxides or protein carbonyls, but once again these are not perfect and not specific for H2O2.

You have a few angles to go for, but just really look into the specificity of the assay before you use it. Too many people use the dyes inappropriately and call oxidation whatever they want to see.... obviously without the proper controls (e.g. antioxidant supplementation or over-expression) this is not correct.

Good luck,

~Adam J. Case

Hi Adam,

you write "You can also look for evidence that H2O2 was present such as lipid peroxides or protein carbonyls" - is there a way to measure specifically lipid peroxides?

The classic measurement of lipid peroxides would be the TBARS assay, but many have shown that the colorimetric assay is flawed and non-specific. Many people use HPLC for TBA, which improves the specificity. Another possibility is to use EPR for lipid peroxides as shown in this paper PMID: 11025200. Of course not everyone has an EPR facility available, nor the technical expertise to do this kind of analysis.

My recommendation when measuring oxidative species is to use multiple methods to validate your conclusion. Use fluorescent dyes, EPR (if you can), and markers of oxidative damage in the presence and absence of specific small molecule or enzyme antioxidants (either over-expressed or exogenously added) to really convince the reviewer but MORE IMPORTANTLY yourself that you are seeing what you think you are seeing.

Unfortunately, our current methods are not perfect, so at this point the more you do the more reliable the results.

Hi,

I am using DHF (Dihydrofluorescein diacetate) for measuring the intracellular ROS generation. The selection of the method to detect specific ROS molecules in different stress are selective and depends on ur interest.

I agree with above answers too and think about that.

Good Luck...!!!

Well, I guess we can say it about majority of molecular biological tools we have - i.e. for demonstration of transcriptional regulation, we use ChIP, EMSA, reporter assays, conferring hypothesis from different angles as Adam truly mentioned above. Same here...

what about tempo-spatial charateristics of H2O2 treatment? i can imagine it would interefere quite fast with intracellular machinery? I do not mean phenotypical changes like death, autophagy, proliferation but rather influence on transcriptional machniery or protein-protein interactions. In other words, could you help me to design an experiment to test a direct ROS influence coming from lipid peroxidations on the function of particular transcription factor?

Hi Adam,

Thanks a lot for your detailed answer; I agree with you that the detection of Intracellular H2O2 is tricky. My interest is to detect both the intracellular and extracellular ROS and H2O2. I am looked for the expression and regulation of NOX4 in human skin cells and interestingly I have got a robust up-regulation of Nox4 after TGF-β stimulation at the RNA and protein level but on the other hand I couldn’t detect ROS production after using the fluorescent dye DCFH and also DHE. Do you have any recommendation or suggestion how to detect ROS production as the only biological activity of NADPH oxidase is the ROS production so I wonder why I couldn’t detect that?

Already appreciable answers are there for ROS measurement. However, I feel non of the answers are pointed towards the original question about using Homovallinic acid (HVA) to measure H2O2 . Additionally, DCFDA is not specific for H2O2. It targets all ROS in general.

HVA is really very very specific for H2O2. 100 uM HVA in presence of 1-2 U horse radish peroxidase (HRP) can be sufficient in 1 ml reaction mixture to standardize the cellular ROS in the homogenate. Dosoki, you can calculate accordingly. But in intact cell it may be difficult to locate only H2O2. However, in intact cells ROS can be measured by FACS using DCFDA. For extra cellular ROS, HVA and HRP system is the best method. You can also try amplex red.

Hi Heba: I was not able to reply your question through mailbox of Researchgate. And I could not find you email so I put my response below. Hope you will find it.

"I use lysate since membrane bound subunit is needed. I keep working buffer on ice due to instability of NADPH."

Hello,

Our group is using the luminol assay to measure ROS in real time. I have read that if you use a dye based assay, including some of the ones mentioned above, and add the catalase/SOD (super oxide dismutase), I believe this would allow you to measure solely intracellular ROS. This approach was mentioned in a couple papers, which I can provide a link to if you want, because the Catalase/SOD can't enter the cell. Thus, when you stimulate/measure ROS with catalase/SOD quenching all ROS outside the cell, you will only detect intracellular ROS.

Good luck with your search!