Our group is using a toxin that we know produces free radicals via activation of nitric oxide synthase-dependent calcium. When measuring other parameters such as lipid peroxidation, GSH and GSSG concentration, the GSH/GSSG also find rate behavior as we expect and this was previously reported (by us and by other groups). But when we measured ROS by this technique, we found that the fluorescence decreases to below baseline. We have hypothesized that the technique fails to measure reactive nitrogen species. Do you agree or think it could be something else?
Let me start by giving this reference which is worth a read if you have even a passing interest in DCFDA: "Measuring reactive oxygen and nitrogen species with fluorescent probes: challenges and limitations" by B Kalyanaraman et al in FRBM 2012 vol 52.
DCFDA is really worth a skeptical look as a ROS-indicating dye because it is not a direct measure of H2O2 and it also redox cycles to auto-amplify signaling. However, it does react with several varieties RNS such as NO2 radical and ONOO. I see no reason why an RNS-heavy cellular system would not light up with DCFDA. Although the biggest fear with DCFDA is the false positive, that apparently is not the problem you are having but it may be as good a time as any to look for another marker of ROS.
In addition to the other problems of self-amplification and an utter lack of R(O/N)S specificity associated with DCFDA, B Kalyanaraman et al put it very well with this quote from the paper I cited: "In addition, one cannot always assume that control and experimental samples exhibit the same efficiency in DCF radical generation and the same linear dependence of self-propagating redox-cycling reactions induced by the DCF radical (Fig. 1)." -- Highlighted mar 14, 2013
Joshua, thank you very much for your valuable contribution in this subject. I defenitely agree with you :)
H2DCF-DA generally measure ROS (H2O2) rather than reactive nitrogen. You can use DAF-2DA, DAF FM DA for nitric oxide and APF for peroxynitrite. Although these dyes are also not very specific to RNS and have been criticized in many recent research papers but still these dyes are in use.
To measure superoxide anion you may try XTT or NBT reduction.
Joshua is right - even although DCF and its derivatives are still quite commonly used, there are multiple problems with it. Though it might be used under carefully controlled conditions as a qualitative indicator, it should be combined with other readouts, and positive and negative controls. The problem is that all of the available techniques for visualising ROS/RNS have serious limitations! Note that DCF was initially proposed as an indicator for NO - but many radicals and oxidants affect it.
Good old NBT method does measure ROS although snsitivity is not so great!
To measure the reactive nitrogen species there will be other probes like DAF-2DA, which is specific for NO. H2DCFDA dye was specifically designed to measure the intracellular ROS. To check your parameters whether the dye working or not/ to standardize the optimum dye concentrations: you can run an experiment with various concentrations of added H2O2. CM-H2DCFDA from Molecular Probes is more potential ROS detector based on my experience. Hope this will help you.
Hi, Daniel, majority of the dyes for ROS are not rathiometric. So, one can not use it for precise quantification of the ROS accumulation. Moreover, DCFDA have a tendency to auto-oxidation in the light, and not precise specific. Similar is true for DAF. Also, you have to take into account that DCFDA can be for intracellular ROS only. have you observe effective dye uptake after treatments? Anyway, you have to insert loading control.
Good luck!
There are electrodes for NO measurements, probabaly it is better way for NO level evaluation in this way than by fluorescence labels using.
Just to add to the above, it is still odd that the DCF signal goes down, whereas the GSH/GSSG and other biochemical indicators go up. How are you performing the experiments? Loading the cells with DCFAM, then visualising them, or or analysing the supernatant? Both techniques are used, but the second requires DCF to be expelled from the cells. The toxin could be either inhibiting the latter or the esterases that convert DCFAM to free DCF. I would do a positive control with say peroxide, with and without the toxin. If the latter reduces the response to peroxide it would suggest that it is interfering with the operation of DCF.
Hi Daniel, DCFDA will react only with one electron oxidants. Namely nitrogen dioxide carbonate radical, hydroxyl radical and compound I of peroxidases (thats why it is used to measure H2O2). Therefore it will measure ROS and RNS. The thing that you NEED to do is used inhibitors such as NAME so the part that comes trough NO and peroxynirite will be diminished by a portion and in this way you can dissect your system. For the record, there is no DIRECT reaction between ONOO or H2O2 with this probe. Only with the derived radicals of ONOO, etc Cheers and good luck
Ideally DCFDA is use to measure total free radical inside the cells, while DAF FMDA is used to measure NO. If you want to measure RNS you can use DAR.
Although these dyes are used to detect reactive species, their specificity is questionable. However you can use DCF DA and DAR to measure ROS and RNS, while using L-NAME you can eliminate majority of reactive species contributed by NO.
Thank you very much to each and every one of you. Yours sincerely Daniel Santamaria
For reactive nitrogen species I suggest the fluorescent probe DAN (2, 3 diaminonaphthalene).
the following link might be useful: http://www.lifetechnologies.com/de/de/home/references/molecular-probes-the-handbook/tables/fluorescence-response-of-3-p-aminophenyl-fluorescein-apf-3-p-hydroxyphenyl-fluorescein-hpf-and-dichlorodihydrofluorescein-diacetate-h2dcfda-to-various-reactive-oxygen-species-ros.html
Whatever probe you apply it is always mandatory to use appropriate controls such as scavengers, inhibitors, alternative detection methods etc. For instance you might use cPTIO as a scavenger of NO or urate as a scavenger of peroxynitrite. An excellent reference for the use of H2DCF in plants is Allan and Fluhr, 1997, Plant Cell, 9: 15559-72. Good speed with your experments!
You can use DAF-2-DA to measure NO intracellular , NO potentiometer to NO efflux quantification, but there is a variety of chemical reactions of NO metabolism generating NO derivatives molecules. So you may quantify peroxinitrite, GSNO, nitrite and nitrate; and
at same time several controls may be done with NO scavanger and NOS inhibitor and SOD inhibitor. I wish all the best wit a lot of experiments
Yes it does mesure RNS. DCFDA is an non specific probe able to detect, for example, peroxynitrite. You can not say what kind of RNS you are detecting without others assays. Since it is non specific do not forget to use NEGATIVE (buffer, SOD, etc) and POSITIVE (Nitric oxide donors, etc) CONTROLS. Best.
but you can check this article
Wang, G., et al., Cell lysis with dimethyl sulphoxide produces stable homogeneous solutions in the dichlorofluorescein oxidative stress assay. Free Radical Research, 2008. 42(5): p. 435-441
Just to add to the discusion, DCFDA is the most popular of the ROS probes, frequently being used to detect ‘cellular peroxides’. However, it is unlikely to do so because it reacts only slowly with H2O2 or lipid peroxides, so for nitrogen species I also suggest the fluorescent probe DAN (2, 3 diaminonaphthalene).
Finally if you want to solve some soubts, I recommend some old, but very classic revies in the field. I am attaching two.
to all
Please, please do read the "Measuring reactive oxygen and nitrogen species with fluorescent probes: challenges and limitations" by B Kalyanaraman et al in FRBM 2012 vol 52. all the answers are there. As the original investigator on the chemistry of DCFH i strongly urge to use it with extreme caution. Please take 15 min and read the article.
Daniel,
It is possible. ROS probe is not a good approach to search RNS. You should check specific probes. Dr. Ischiropoulos suggested a good reference abou free radical probes.
Good luck
Fluorescent redox probes are notorious for artifacts and are nearly meaningless. The misinterpretation of fluorescent redox probe data has cast a pall of doubt on the entire field of free radical studies. I suggest using chemical methods and only using fluorescent probes once you have established that oxidative stress is occurring through other means.
