Hi,
I want to measure ROS activity in the treated cells.I would prefer a way that is more trustful. For this case, I can measure concentration of different ROS produced in the cells under treatment. However, decided to measure extracellular H202 using by Amplex red. Do you think that if I use this method only to speak about ROS production, it can be enough.
Any suggestion would be appreciated,
thanks
Intracellular reactive oxygen species (ROS) was estimated by
using thefluorescent probe, DCFH-DA as described earlier by
Ramful et al. (2010). Briefly, the cells after preincubation with
different concentrations of the extracts (1, 10 and 50mg/mL)
for 24 h were treated with tertiary butyl hydrogen peroxide
(100mM) for 15 min. After washing with phosphate buffer
saline (PBS, pH-7.4) cells were treated with DCFH-DA (20μM)
for 30 min. After washing with PBS, the cells were imaged
using confocal microscopy (BD Pathway 855, BD Bioscience,
USA) equipped withfilters in the FITC range (i.e. excitation,
490 nm; and emission, 525 nm).
see the attached article
I suppose, You may have problems with any method, dependently on reviewer. DCFDA is lesser selective than Amplex Red, however, you may be asked to measure ROS with Mito-SOX, dihydroethidium (+ HPLC), or anything else. If I measured ROS in cells I would use both DCFDA and Amplex Red, and, in addition, determined one related parameter - GSH/GSSG ratio, for instance.
Dear Mahbobeh
I suggest to do more than one protocol of ROS analysis in treated cells this will give a comparative report and will be more authentic data.
I think GSH/GSSG ; DPPH ; DCFH-DA are the most common protocols for ROS analysis depending on compatibility with your system you can choose at least two of these protocols.
Hope it answers the question
First, you need to ask yourself the correct question.
"How to measure ROS activity" is not correct question. ROS is an abbreviation and "abbreviation" does not have biological activity. Free Radic Biol Med. 2015 Jan;78:233-5.
Please specify the particular species to study, for example, superoxide, hydrogen peroxide or peroxynitrite. Note that there is no any probe or process which detects all of these species. These species also act by distinct biological pathways. Free Radic Biol Med. 2008 Nov 1;45(9):1340-51; Antioxid Redox Signal. 2014 Jan 10;20(2):281-94.
Second, once you defined your question correctly, for example, "How to measure hydrogen peroxide production and biological activity" then you can identify correct methods and protocols. You can detect intracellular H2O2 by boronate probes and define H2O2 biological activity by treatment with H2O2 scavengers such as PEG-catalase (200 U/ml, 4-24 hours). Antioxid Redox Signal. 2014 Jan 10;20(2):372-82.
Some methods and probes should not be used despite frequent utilization in literature, for example. lucigenin, DCF-DA and DHR. Free Radic Biol Med. 2012 Jan 1;52(1):1-6.
For references please see my Research Gate page: https://www.researchgate.net/profile/Sergey_Dikalov/publications
Sergey.
Sergey Dikalov gave a very good comment. I can only add to this that when you plan to study ROS production by cultured cells you have to be not only specific, as Sergey advised, but also remember, that the cells grown in the presence antibiotics are not suitable for this purpose. Some substrates present in the culture or incubation media, namely pyruvate and other ketoacids, nonenzymatically react with hydrogen peroxide.
Finally, water and solutions themselves may be highly contaminated with hydrogen peroxide, particularly water from milliQ systems. H2O2 may also enter the media from air if you live in a South or use instrument not equipped with the system,. which removes ozone formed by the light source.
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