Hello,
I would like to ask whether anybody has any experience in methods detecting intracellular ROS. Which method would you suggest for detecting ROS both in cell cultures and tissue samples?
The most commonly used reagent for measuring oxidative stress is H2-DCFDA (measures H2O2). If you want to measure superoxide, use HE. If you want to measure mitochondrial superoxide, use Mitosox. What do you mean by tissue samples? If you want to measure ROS in vivo, use luminol or L-012. There are protocols out there for all of these.
thank you, I know about all the in vitro assays. By saying tissue samples, I mean in vivo-in situ detection (biopsy specimens). I am interested generally in oxidative stress, not specifically for H202 or mitochondrial origin.
There is no perfect way to measure intracellular ROS in biopsy specimens (if intact). If you have microscope slides or whole tissues, you would have to measure oxidative stress markers (See linked paper below). You could also look at the expression of antioxidant enzymes. However these would all be indirect measurements of oxidative stress. You could use the same techniques on cell cultures (in vitro) as well.
Article Detecting Reactive Oxygen Species by Immunohistochemistry
There are some reports concerning DHE assay performed in OCT-frozen tissue sections; I am not so optimistic though, do you have any experience on that?
I don't have experience measuring ROS in tissue sections. Most of what I have done is in vitro and in vivo (mice, bio-imaging). In my experience, measuring ROS with any degree of confidence is tricky, given how unstable they are. I looked up what you mentioned (I too am highly sceptical of this). See the link below for further discussion on this topic.
https://www.researchgate.net/post/How_long_can_I_store_OCT_embedded_tissue_to_still_be_able_to_get_valid_measurements_for_ROS_using_DHE_staining
I would say your best bet would be to measure oxidation/peroxidation products, rather than trying to measure ROS. Below is a paper that I read a while ago where they were trying this. Could give you a general idea of the assays (Keep in mind that there are kits available for most assays these days. Cayman has some good ones and the following guide for picking the right oxidative stress markers).
Article Effect of Salmonella enteric Serovar Typhimurium in Pregnant...
https://www.caymanchem.com/cms/caymanchem/LiteratureCMS/800195.pdf
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