Actually on the stress condition cells produce ROS and RNS that ultimately leads to cellular oxidative stress. HOW can detect RNS and superoxides
For superoxide detection XTT working pretty well (see 1st link). And please follow 2nd link to find our protocol for RNS metabolites determination.
https://www.researchgate.net/post/How_do_I_measure_nitric_oxide_measurement_in_spinal_cord_tissue#561f7e845cd9e317518b4586
Conference Paper THE PECULIARITIES OF EVALUATION OF OXIDATIVE METABOLISM IN C...
XTT protocol:
100 ul cell suspension in HBSS per well of 96-well plates
1 hr 5% CO2, 37ºC
100 ul warmed XTT 125 мкM (0,083 mg/ml) in HBSS (PMA or Zymosan A optional as a test agent or positive control for ROS production in phagocytes)
1 hr 5% CO2, 37ºC
measure OD 450 nm with reference at 650 nm.
Thanks dear Daniil R. Petrenyov , can you suggest any fluorescence based assay for detection of Reactive nitrogen species (RNS) and superoxide?
it depends of your aim and experiment follow link to select suitable for you
for RNS read an article attached
http://www.thermofisher.com/order/catalog/en/US/adirect/lt?cmd=catDisplayStyle&catKey=101&filterDispName=Free+Radical+Detection&filterType=1&OP=filter&filter=ft_1701%2Ff_219301*&_bcs_=H4sIAAAAAAAAAH2MQQqAMAwEX5OTKFpBvHrxLPiEGqlgjaSp%2Fb7rB4RlB2Zh647acVHZsrdUkRuq%0AlfU5PKcfH8xu6idyM1JKaSywRtmPBDZeInROKL5QQSIDohvGYPHEA7n%2BSzuaZgZedPbeBIcAAAA%3D
https://www.thermofisher.com/by/en/home/references/molecular-probes-the-handbook/probes-for-reactive-oxygen-species-including-nitric-oxide/generating-and-detecting-reactive-oxygen-species.html
Article Improved Methods to Measure End Products of Nitric Oxide in ...
Commercially available tools for RNS related assays include:
(1) DAF-FM diacetate, a fluorescent probe for NO. You can do FACS, 96-well, imaging with it.
Please this older thread:
https://www.researchgate.net/post/Does_anyone_use_flow_cytometry_for_nitric_oxide_NO_detection
(2) Griess reagent, measuring nitrite as a footprint of NO. A cumulative assay.
(3) anti-3-nitrotyrosine antibody, for assessing the level of protein ntiration, as a footprint of peroxynitrite. But substantial works show that nitration can be HOCl mediated. Avoid using DTT and similar reducing agents in Western blots.
(4) if you are doing an inflammation model, you can check also iNOS expression level, or phospho status of eNOS/nNOS.
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For superoxide, you can try dihydroethidine (HE) or its mitochondrial version, MitoSOX. The probes are not very suitable for imaging because of a known artifact from overdose or prolonged staining, DNA binding which exaggerates the fluorescence. < 5 uM and 15 min leading for MitoSOX is recommended.
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