Hi together,

does anyone have a detailled protocol for the quantification of ROS in an electrolyte solution, i.e. in the absence of any cells?

I am currently working on this with the fluorescent dye Dichlorodihydrofluorescein diacetate (DCHF-DA; C24H16Cl2O7)

https://www.sigmaaldrich.com/DE/de/product/mm/287810

and the following protocol:

https://pubs.rsc.org/en/content/articlelanding/2018/en/c8en00055g

However, I am not yet happy with the results. A standard curve with H2O2 (0.1 µM to 12 mM) only had a very flat slope and very low absolute fluorescence values. Comparisons with other protocols showed that DCHF-DA usually is hydrolysed to DCHF by 0.01 N NaOH (this is described differently in the protocol above), a step which is usually done by cells when involved in the assay. The resulting DCHF is non-fluorescent and sensitive to oxidation, so that the presence of ROS can transform it to Dichlorofluorescein (DCF, without dihydro), which is fluorescent and, thereby, can be quantified spectrophotometrically. I could imagine that in my case only a small fraction of DCHF-DA is hydrolysed to DCHF, maybe due to a too low concentration of NaOH. However, I also found other protocols that worked with 0.01N NaOH.

If you have any helpful protocols or ideas, please let me know!

Thanks and best wishes,

Jonas