I wish to estimate ROS production using U937 cells. I have found some papers using Lucigenin for the chemiluminescent assay and some papers using DCFH-DA. Can you please let me know a solution for this and how to go about?
Thank you. :)
I used both of them but due to fluorometric assay of DCFDA it is more sensitive than lucigenin or luminol by the way.
Article Hydrogen peroxide: A potent inducer of differentiation of hu...
there are several ways for determining ROS production . DCFDA is the most sensitive, valid and known way.
Theoretically, luminescence is way more sensitive than fluorescence. In practice, however, it is dependent on the instrument. If you have an access to a good luminometer than you may try lucigenin. Otherwise use fluorescence-based assay. Indeed, any fluorometer can be used to detect luminescence but they are not specifically designed to do it therefore fluorescence detection is more suitable/reliable.
Denied. Even in theory the magnitude of fluorescence agent is over the luminescence.
https://www.highveld.com/pcr/fluorescence-chemiluminescence-6232.html
Article A comparison of fluorescence versus chemiluminescence detect...
Chapter Overview of Colorimetric, Chemiluminometric, and Fluorimetri...
Do you have a specific ROS you want to measure, or just overall oxidation products? Dichlorofluorescein is non-specific and prone to artifactual amplification by a number of reactions, I suggest reading this paper to get an idea of a number of ways to measure different ROS, with their associated pros and cons:
Article Measuring reactive oxygen and nitrogen species with fluoresc...
I completely agree with Christofer, DCFDA, is not very specific and dependent form intracellular esterase/proteases activities. For the superoxide you can use DHE what is more specific. Moreover, one not measure ROS production, but only ROS accumulation. Because in each cells occurred simultaneously ROS generation and scavenging. One must be carefully with terminology.
My best wishes!
Taras Pasternak Thank you. I am doing the optimization experiment using U937 cells against Cancer cell. Hence I have used the term ROS production. I am not quite sure of it.Please do rectify me.
Christopher Paul Hedges I am not quite sure but the species produced by monocytes . So, please suggest me a way. Thank you for your reply.
Also some papers say DCFDA, some DCFHDA. So I need to know specifically which one to use.
Thank you for the help everyone.
Dear Gargi, actaully one can measure only ROS accumulation ea differences between production and scavenger. However, this definition is dependent from method you used.
cells have evolved multiple systems to control ROS levels and always exist a balance between generation and de-toxification.
Pure production you can measure only if you completely shut down all scavening system, but it is not a case,
I would suggest to mention ROS accumulation, what is much more correct.
Have a nice day!
I appreciate answers from everyone, in particular that of Christopher. But, I feel that it is not appropriate to use "ROS accumulation". ROS are short-living and accordingly Dr. Pasternak has categorically and aptly stated that there is balance between generation and detoxification. Being highly reactive, reactive oxygen species would interact with almost every cellular component [lipids, proteins, nucleic acids etc.]. They instantaneously interact and indeed get detoxified by several non-enzymatic antioxidants [like - range of phenolic compounds, vitamins like ascorbic acid, tocopherol, polyols, pigments etc.], besides range of enzymatic antioxidants. Coming back to the actual query, to the best of my knowledge there is no ideal/perfect protocol for determining ROS production. All the known protocols [irrespective of use of DCFDA, lucigenin, luminol or something else] rely on basic redox reactions. Therefore, there would invariably an error [however, we can certainly talk about variation in relative ROS production between treatments]. I still believe that lipid peroxidation protocol [measurement of MDA production], remains relatively better for measuring ROS production.
Dear P. Pardha-Saradhi , I agree with you,it is better to tell ROS balance ea. differences between production and scavenger. Lipid peroxidation occurred only under excess ROS accumulation and cant serve as a marker of production. ROS continuously produced, but peroxidation occurred only when balance between production and scavenging are disturbing. DSHBS/AAP method is very good for measure such balance. Please, look here: https://www.cell.com/trends/plant-science/fulltext/S1360-1385(16)30112-1
My best wishes!
Dear Dr. Pasternak,
Thanks for your nice comment. I do agree that lipid peroxidation test also can't be ideal marker of ROS production. This is a highly debatable area.
best regards
Thanks!
Lipid peroxidation just reflected excess ROS what is not scavenging by scavenger in cells. But this excess does not rekated with production, for example, mutant for ROS scavenger may increasing peroxidation even without increasing in ROS generation.
ROS generation cant be measure in real situation because of simultaneous production and scavenging. In theory, you can measure production if you stop all scavenger. But this is impossible, +My best wishes!
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