We employ aortic ring assays to study effects of specific gene knock-outs on endothelial sprouting. The assay itself works nicely; control ring sections respond to VEGF in the expected fashion. Now we want to test if the observed effect in one of our mouse models depends on oxidative stress. To reduce ROS, we cocultured rings in N-acetylcystein. Is this the optimal compound? At what concentration did you use it? What could be alternatives?
Hi, we used Idebenone (1 mmol/L) in our experiments [Mordente A, Martorana GE, Minotti G, Giardina B. Antioxidant properties of 2,3-dimethoxy-5-methyl-6-(10-hydroxydecyl)-1,4-benzoquinone (idebenone). Chem Res Toxicol. 1998;11:54–63.] NAC somtimes had unspecific effects.
superoxide dismutase mimetic (Mn-III-tetrakis-4-benzoic acid-porphyrin; MnTBAP) might be considered as in a recent study published in journal of vascular surgery 2012;Volume 56, Issue 5 , Pages 1381-1389.
pegSOD and pegCat could be used as specific cell-permeable ROS scavengers, you could wait the effect about 24 hours after administration.
Why not try what is actually used in vivo to control oxidative stress in the relevant setting? These are mixtures of biomolecules naturally present in tissues. These can be isolated from any tissue, and likely function in an emergent manner to control ROS toxicity and function. We only know a few of the systems (SOD, catalase, glutathione....). What makes you think adding an artificial compound into an artificial assay system (the ring assay) has any meaning to development of advanced compounds, or understanding of angiogenic activities in vivo? All the single factor blockers of angiogenic activity have limited utility. Natural emergent mixtures are the "game-changer" that is needed in this field. I would be happy to discuss methods of isolating such useful mixtures from human tissues if anyone is interested.
THANKS for all your answers. 'Will try some of the compounds, then we'll see what happens...
I recently published an article on ROS inhibition using NAC 10mM but I got to argue with one reviewer about its specificity. Some authors say it inhibits NFkB, others say that it is because it inhibit ROS, which was suppose to dissociate NFkB from its inhibitory subunit IkB ... To avoid problem later, I suggest you use 2 antixoxydants and show that they reproduce the same effect.
Thanks, Bryan. I will use NAC and Tetrakis and hope for pleased reviewers.
Most people will not like NAC. Is not specific and has effects in multiple ROS and inflammatory systems. It would be better to use an SOD or catalase mimetic such as Tetrakis or Tempo. Ideally you would like to block the source of ROS with a broad inhibitor such as DPI or an inhibitor for the specific oxidase.
Dear Sven,
If you like, you can have a look at our recent review articel covering that topic:
Article Functions of ROS in Macrophages and Antimicrobial Immunity
In this review, we give an introduction to ROS and their sources in macrophages, summarize the versatile roles of ROS in direct and indirect antimicrobial immune defense and provide an overview of commonly used ROS probes, ROS source inhibitors and ROS scavengers (also the difference between ROS scavengers and antioxidants, which are not synonymous, is explained).
If you have any further question please ask anytime.
All the best and stay healthy,
Marc
Resveratrol interacts with ROS as an antioxidant. It affects the expression and activities of ROS-producing biomolecules. The effects of resveratrol on cell culture can be tested.
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