I am looking forward to perform ROS imaging in Arabidopsis thaliana roots using H2DCFDA. I am not sure which solvent should I use for dissolving DCFDA and what should be the ideal concentration for best results?
Hi,
You should check the recommendations of the company from which you bought it. Usually the best solubility is in DMSO, so you can prepare the stock solution in it (25-30mg/ml). Your working concentration is probably sufficiently low for it to be soluble in an aqueous buffer.
H2DCF-DA can be dissolved in HBSS. In my cell line, I incubated cells with 100uM for 1 hour at 37 deg C. You might want to test different concentrations starting with the manufacurer's recommendations.
Yes, DCFDA stock is solvable in DMSO and the staining method is very simple: just immerse root in 20 micromolar DCFH-DA for 15-30 min- you should figure out the right time: see our paper (it's for Vicia, but it works equally well for Arabidopsis in our lab)- Garda et al., 2018, Front. Plant Sci. 9/1823
Why i get high intensity in untreated plus dcfda than positive control and other treatments?
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