Dear Julian; independent on what are the best conditions to induce oxidative stress, as other researches have mentioned, DHE is detecting the presence of ROS in your system, and it can very well be that the ROS were present but at the time of your measurements they are reacted and undetectable. Be carefull when using H2DCFDA, H2O2 in the presence of peroxidases, that all cells have would give you a strong signal (I do not have experinece with DHE in that sense. instead 8oxodG is an index of DNA damage by oxidative stress. The presence of ROS does not imply oxidative damage. So the question is what you want to measure.

How you apply the various concentrations of H2O2 (200uM, 400uM, 600uM and 800uM) within the four-hour treatment? Some papers/protocols suggest cultured cells to be exposed to H2O2 at 37°C for 15 min at indicated concentrations and immediately washed three times with cold PBS. For serial treatments, different amounts of H2O2 (0.1–0.6 mM) could added to the culture medium every hour without changing the medium. At 15 min after the final treatment, the cells should be washed with PBS, scraped, centrifuged, and stored at –80°C until use. Can you share more details about your H2O2 cell treatment protocol?

The best results we have gotten was using DCFH in flow cytometry. Due to the oxidative stress responding dyes have very close exitation and emission spectra, they require instant measuring to get certain detection levels, which might be varying due to treatments. H2O2 can not be a best choise I assume, however, you should be able to measure the difference in between controls and treatments using DCFH dye incorporated to flow cytometry.

The concentration of H2O2 is pretty high and under such conditions cells may be serverely damaged. So, I would have two recommendations: change both, the inducer and the reporter. As for the latter, we often used CM-H2DCFDA as fluorescence probe, which is suitable for microscopy, FACS, microplate assays etc, or we used L012 for chemiluminescence assays. Both are sensitive and work fine in our hands. For more information, go to the Invitrogen homepage, choose Molecular Probes and check the Mol Probes Handbook, which you'll find online and free. There you choose the ox stress chapter and will find dozens of reporter systems, so may may choose what suits your application and devices. As for the inducer, H2O2 didnt work good in our assays either. Besides, you damage cells and mitochondria will respond by producing even more ROS. We used DMNQ, pyocyanin, paraquat, and whatnot to induce ROS. You'll also find a lot of details in the Mol Probes handbook. Follow this link:

http://de-de.invitrogen.com/site/de/de/home/References/Molecular-Probes-The-Handbook.html

Good luck.

We used H2DCFDA as intracellular probe for ROS which worked reasonably well with H2O2 levels < 100 µM. We never tried DHE...

We recently started to measure 8-oxo-dG levels using the EIA from Cayman and the OxyDNA kit from Argutus. The assays works very well, however me made a startling and somewhat uncomfortable discovery. As you have observed with your cells, there was no significant difference between H2O2-treated and untreated cells. This is most likely due to the fact that we (which means most researchers) culture our cells at 21% oxygen. However, inside tissues the O2 partial pressure is more close to 2-4% O2, which means that our cell lines are already having maximum oxidative stress from the environmental O2 which cannot be further increased by adding H2O2. We are currently establishing a normixia/hypoxia culture (5-1% O2) to test this hypothesis (which is supported by Argutus, the manufacturer of the OxyDNA kit). I can let you know, but it will take at least 2 weeks before we have the results.

You should also be aware that H2O2 alone is no strong DNA damaging agent, you need to add Fe2+ (Fenton reaction) to get DNA adducts.

I propose to load the cells with esterified H2DCFDA as intracelluar probe. We have obtained good results with this probe . Another, problem is that added H2O2 decomposes ore or less rapidly in medium. You should check how much H2O2 is present after 10, 20 30 min..by means of Amplex Red/HRP. Alternatively, you may apply paraquate to initiate intracellular ROS level.

Dear Julian; independent on what are the best conditions to induce oxidative stress, as other researches have mentioned, DHE is detecting the presence of ROS in your system, and it can very well be that the ROS were present but at the time of your measurements they are reacted and undetectable. Be carefull when using H2DCFDA, H2O2 in the presence of peroxidases, that all cells have would give you a strong signal (I do not have experinece with DHE in that sense. instead 8oxodG is an index of DNA damage by oxidative stress. The presence of ROS does not imply oxidative damage. So the question is what you want to measure.

Hi, Juliana, have you tested cell viability after 4 hours treatments with 800 µM H2O2? Have you used new DHE: DHE is quite unstable and can subjected to degradation even at -20°C. Which stage of the cell development have you used? You will detect ROS accumulations rather in the later exponential phase, at the early stages cells have a very effective mechanism of ROS scavenger.

You can also try other ROS-induce agents like menadione or paraquat: both can directly induced superoxide. but external H2O2 does not directly induced superoxide.

You also can try H2CDFDA, but in that case you will never now H2O2 source, you need to treated cell with other ROS-induced agents. Because almost all dyes is not ratio-metric, you have to use a control for dye uptake and distribution. Goog luck!

Julian, As mentioned before H2O2 concentrations are too high to be able to see dose dependence. With lower doses and 15 min treatment (followed by washing) you should see the difference. DCFDA, even if it is widely used, does not measure ROS and is not recommended for research (http://www.sciencedirect.com/science/article/pii/S0891584911006058).

Pls see our paper,might be helpful in adjusting protocol (https://www.researchgate.net/publication/233797603_Rapid_and_Specific_Measurements_of_Superoxide_Using_Fluorescence_Spectroscopy?ev=prf_pub)

Article Rapid and Specific Measurements of Superoxide Using Fluoresc...

Juliana, as mentioned by several others the concentrations of H2O2 you are using are extremely high. We have successfuly measured oxidation of DCFHDA, pHyper, mito-roGFP, p47-roGFP, roGFP2 with H2O2 concentrations in the low micromolar, which is closer to physiolgocal H2O2 concentrations. I think Juan Reyes asked a very important question, "what do you want to measure?" Answering this will allow you to develop an appropriate protocol.

Hi Juliana. As other colleagues have pointed out, H2O2 are maybe too high for your system and you may have just dead, unresponsive cells. Also, it would be advisable to measure the linearity of the response of the indicator dye to the selected H2O2 concentrations in vitro to ensure you will measure concentration-dependent outcomes. It is also important to consider the type of cell culture you are using, since their resistance to oxidative stress (i.e. their level of intracellular antioxidants) may be quite different. In my experience, it is very difficult to extrapolate results from cell lines (quite resistant to oxidative stress). If you are not employing primary cell cultures, I would suggest to do so. Good luck!

The main question is what you want to measure: the superoxide levels or the effects of this oxidative insult on molecules such as DNA (8-OHdG), lipids or proteins.

The other problem is that there isn't hydrogen peroxide anymore four hours after H2O2 treatment : it is dismutated to water and O2 spontaneously or after transmembrane diffusion through aquaporin channels it is scanveged by endocellular antioxidant enzymes so it will be difficult to detect it or its derivated compounds (OH radical produced by Fenton reaction). Also even if many studies have documented increased O2.- and increased cytotoxicity in different in vitro cellular models exposed to H2O2, this radical specie has a half-life very short because is very instable and also is promptly eliminated...taking in to account these observations in my opinion the treatment duration is very long; moreover you use H2O2 concentration too high, I haven't ever read about 800 microM..perhaps until to 500. Anyway, when I detect the ROS levels I usually use the DCF-DA (2',7'-dichlorofluorescein diacetate) method on cells treated with H2O2 (200-300 microM) for 20'-30' and then a ROS measurement is immediately taken. Equally I treat the cells when I evaluate the oxidative DNA damage as 8-OH dG by Comet Assay. In this case the H2O2 treatment produces an increase of oxidative DNA damage that is highest 15-30' after removing H2O2.

Thus I highly reccomend you to modify your experimental protocol and repeat your measure before changing the methods used.

Hi, Juliana, please, also consider that main source of intracellular superoxde is mitochondrial electron transport chain. External H2O2 have a very liitle effect on superoxide, one of them may be through inhibition of some types of SOD. As additional control you should have a viability test, for example through FDA. Moreover, FDA can serve as a positive control for 2',7'-dichlorofluorescein diacetate, if you plan to use this dye as well. This control is crucial if you want to have quantitative data, because 2',7'-dichlorofluorescein diacetate is not ratio-metric dye and signal strongly dependent from dye uptake capacity. In addition, you can measure the H2O2 level in the medium by DCHBS/AAP method (but you should use medium without phenol-red, because phenol-red may interact with the measurements).

Good luck!

Thank you everyone for contributing ideas. I really appreciate your effort..

My aim is to compare the level of ROS in H2O2 treatment alone in endothelial cells versus to level of ROS in endothelial cells pre-treated with drugs (anti-oxidant) prior to H2O2 insults. Correct me if I'm wrong, from my understanding, level of ROS in cells is correlates to the level of oxidative stress in cells right?

From my MTT cells viability data (taken after 4 hr H2O2 treatment): 800uM H2O2 for 4 hour kills the cells, however, 400uM, 600uM and 200uM H2O2 for 4 hours do not effect cells viability as compared to untreated cells. I'm testing out 800uM H2O2 because I just want to make sure that our H2O2 30% stock is still working.

From my ethidium fluorescence intensities data, 200uM, 400uM and 600uM H2O2 treatment (4hr-treatment) in endothelial cells does not have any significant dose-dependent in the level of ethidium fluorescence intensities. Therefore, I'm wondering what's gone wrong.. Is it the reporter dye is not suitable or the H2O2 even at 600uM (4hr-treatment) is still not enough to induced significant oxidative stress in cells or the H2O2 is already converted into something else and no longer functioning in the media throughout the treatment.

Basically, my treatment protocol for H2O2 treatment in endothelial cells is listed below:

1) Seed cells in 96-well plate. Incubate overnight-24hour.

2) Add compounds of interest. Incubate for 24hour

3) I'll remove the conditioned media, and replace with complete ECM media (FBS +PenStrep +ECGS) + H2O2 various concentrations: (200uM, 400uM, 600uM) and incubate for 4hour at 37C, 5%CO2 incubator

3) Add DHE into each well. Incubate at 37C, 5% CO2 incubator for 30min.

4) FIx and wash cells once with 1xPBS

5) analyze fluorescence intensities in the nuclear.

Therefore, anyone here has experience in using H2O2 to induced oxidative stress in endothelial cells, please leave some comments on my protocol / methods.

I think you should add DHE before adding H2O2. After 4 hrs, H2O2 will already be degraded...

How do you fix the cells? Are you shure your fixation method does not interfere with the DHE dye? Why don't you measure live cells?

Hi, Juliana,

Intracellular ROS level may not correlated with oxidative stress, in many case extracellular ROS have much stronger impact on the cells and can be consider as oxidative stress marker. You can use DCHBS/AAP method for evaluation of the ROS level.

Intracellular superoxide usually related mainly with mitochondria electron transport chain, but with PM.

Please, consider that all NOX generate extracellular ROS, which can not be detected by DHE.

Moreover, you should not fix cells, with fluorescent dyes you should use only living one.

For your experiment, I would suggest to evaluate ROS level not only after 24 hours, but after short periods.

If you have plate reader, you can add your compound of interest directly to well (after 24 hours pre-incubation), add DHE (or other fluorescent dyes) after 4 hours incubation to the same well and start to measure by taking at least 3-4 time points in real time.

This will save your time and you will get dynamical changes in the ROS level after addition of your compounds if interest.

Please, also do not forget include control for dye uptake and cell viability (FDA, for example).

Good luck!

Try using amplexred, it works great in cultured cells. In order to complement your "redox status" do ROS measurements (DCFDA, DHE) together with GSH/GSSG ratios

Sorry to disagrre with Dr's Nazarewicz last statement, that maybe came out short of words. H2DCFDA does measure ROS and RNS, specially under certain circumstances that are normally found in cells. Besides, Kalyanaraman et al, in their review do not say H2DCFDA should not be used for research. That general statement cannot be said for any compound or subject in a free world. Certainly, the complexities of H2DCFDA cell chemistry call for a cautious interpretation of the results. However, undoubtedly, redox processes are involved, and probe oxidation is a result. Furthermore, do the other probes measure only ROS? The same work by Kalyanaraman et al suggest that no. So, cautiuos in result interpretation is always a must. And more than one probe is highly recomended, and might even suggest mechanisms for the phenomena observed.

Hi, Juan, you can use H2DCFDA for the localization of the H2O2 accumulations. I am not sure you can make precise measure because this is not a ratiometric dye. If you insert control for dye uptake, you can tell about ROS level. For precise measure one should use only ratiometric dye. Good luck!

Hi Juliana, If you are concerned that your H2O2 stock has degraded you can check the concentration by using absorbance spectrophotometry. Measure the absorbance at 240 nm of a diluted (try 1:25 - 1:100) solution of your H2O2 stock. You will need to use an appropriate plate or cuvette that is UV transparent. Apply beer-lambert law using extinction coefficient of 43.6 M-1 cm-1 and calculate concentration. Remember to adjust the calculation for path-length if you use a microplate.

Do you have a way to measure the peroxide in your medium? Most cells have peroxidases and catalase that dispose of peroxides. Depending on the volume of media and cell number most of your peroxide may be gone in an hour and the cells well on their way to recovery at four hours.

Hi Juliana, you may use 2',7'-diclorofluorescein diacetate, with this compound you are able to detect early ROS production, in my experience this compound is more sensitive than DHE.