Please refer to following link: https://bio-protocol.org/e2545

I am sure Prof. Sinha or Prof. Singh will help you in case of further questions.

I would go with DCF-DA. Given that it is converted to its non-fluorescent form once inside the cells, it is a better indicator of real time changes in cellular ROS levels. If you intend to measure changes in ROS in response to treatments, this becomes even more important.

Just go with DCF-DA / H2DCFDA / DCFH-DA. They are basically the same non-fluorescent probe which will turn to highly fluorescent DCF upon oxidation.

Thank you all for your kind suggestions. May I please know the working concentration of DCFH-DA??? Am I supposed to wash the cells with PBS before taking it for flow cytometry???

Usually 1-10 uM. You need to use serum free medium for staining, wash the cells with PBS and add phenol free medium prior to acquisition. Check this link for best practices:

https://assets.thermofisher.com/TFS-Assets/LSG/manuals/mp36103.pdf

Pls. check this link; Article Detection of reactive oxygen species (ROS) by the oxidant-se...

Hey Sheelu,

as mentioned before there is indeed not much difference between these two substances. However, we and others compared 2′,7′-Dichlorofluorescein (DCF) and 2′,7′-Dichlorofluorescein diactetate (DCF-DA) with its derivate 6-carboxy-2,7-dichlorodihydrofluorescein diacetate, di(acetoxymethyl ester) and while the two commonly used DCF derivates DCF and DCF-DA freely diffuse over membranes (so into organelles and out of the cell) the latter derivate stays in the cytosol. If you want to measure "overall ROS" in the cell it does not matter, but if you want to detect cystotolic ROS you should try the 6-Carboxy-Derivate.

For further information and details for ROS measurement techniques please have a look at:

Mitochondrial reactive oxygen species enable proinflammatory signaling through disulfide linkage of NEMO, Science Signaling (2019)

and

S. L. Hempel, G. R. Buettner, Y. Q. O’Malley, D. A. Wessels, D. M. Flaherty,

Dihydrofluorescein diacetate is superior for detecting intracellular oxidants: Comparison with 2′,7′-dichlorodihydrofluorescein diacetate, 5(and 6)-carboxy-2′,7′-

dichlorodihydrofluorescein diacetate, and dihydrorhodamine 123. Free Radic. Biol. Med. 27, 146–159 (1999).

All the best,

Marc