Because one of our drug is fluorescent active we are not able to detect ROS through florescent detection method.
Hi! With relation to sensitivity and specificity fluorescent approaches are superior. If fluorescence is not viable (as in your case) you may have recourse to luminescence, eg. HRP luminol (for H2O2) or lucigenin (for superoxide). Both methods are relatively resistant toward interferences. Years ago (when I had no access to fluorimeters or luminometers) I have performed in situ detection of ROS (most likely superoxide anions) by reduction of nitro blue tetrazolium, which leads to the formation of deeply blue formazan aggregates. These, in turn can be detected microscopically or solubilized to perform quantitative measurements. Extinction in the blue light range seems to correlate with oxidative stress. I hope, that my littele suggestion is of value for you.
Good luck!
ROS measurements by lucigenin (for superoxide) and by HRP-luminol (for peroxide) is the standard one. You can also measure superoxide levels by HPLC analysis by using specific probe such as MItosox for mitochondrial superoxide and DHE for extra-mitochondrial superoxide levels.
Which exitationa nd emission your drug have ? Ther are a couple of dluoresent dyes with different wavelength. You can choose ROS senitive marker with the other fluoreesnt values.
We measure ROS (hydrogen peroxide) using a luminometer (HRP and luminol). This seems to be an accurate way of measuring, because we also performed simultaneous detection of ROS by fluorescence and the trends were relatively the same.
Hi Saswat, If you have Flow cytometry facility in your Institute, Please use DCFDA or CMH2DCFDA for ROS measurement in cancer cells. If you need help, I can extend. If your drug is fluorescent active, just avoid that channel of fluorescence for selecting probe. Thanks.
Alak
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