I want to detect ROS and NO generation in primary macrophages. DCF-DA and DAF-FM are the methods used by my lab for ROS and NO detection by Fluorimetric analysis. They do this by trypsinizing the cells followed by fluorometric analysis. I don't want to give stress to macrophages by trypsinization. Is their any method to detect ROS and NO by IHC. Any other method is also appreciated.
Dear Durgesh,
Without trypsinisation also you can do those experiments by using fluorescence microscope. Also you can check the protein levels of cytochrome-C by WB or IHC. Cyt-C is corelated with ROS generation. ROS can lead to apoptosis. It may go through the p53 pathway also.
Hello, you can opt for a microscopy approach for measuring DCF fluorescence intensity (preferably through LSCM). Please have a look at Fig. 2 of our recent publication. Hope this helps.
Article Mitochondrial Oxidative Stress-Induced Epigenetic Modificati...
Please check my paper " potential theranostics applications of biosynthesized siver nanoparticles".
HI, You can check for inox, enox, nnox , hsp70 protein expression by ihc for free radical production
ROS can be measured by using fluorescence microplate reader without the need of trypsinization. I have used this personally and it gives good results using DCFDA. NO can also be measured in culture supernatant using DAN method, again by fluorescence plate reader. Please read the following papers. I hope this helps.
Wang H1, Joseph JA. Quantifying cellular oxidative stress by dichlorofluorescein assay using microplate reader. Free Radic Biol Med. 1999 Sep;27(5-6):612-6.
Misko TP1, Schilling RJ, Salvemini D, Moore WM, Currie MG. A fluorometric assay for the measurement of nitrite in biological samples. Anal Biochem. 1993 Oct;214(1):11-6.
I am struggling with similar issue. without trypsinization u can do microscopy or you can measure secreted ROS (H2O2). For microscopy you can use any ROS sensitive dye , DHE, Mitosox, DCF. For secreted I have used amplex red. All this will depend on the strength of ROS signal you are expecting.
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