Could somebody please explain why I'm obtaining fluorescence signal when I combine DCFDA and H2O2 in buffer? My understanding is that DCFDA must first react with esterases to eliminate the acetyl groups, and that's why DCFDA has to be used with live cells. So, why I'm getting this fluorescence signal? I tried with different H2O2 concentrations, and the signal was proportional with the increment of concentration. Is it because the binding with the acetyl groups is not stable in buffer?
Thank you!
Dear Zavatti,
Several one-electron-oxidizing species will oxidize DCFH to DCF. These include hydroxyl radicals (•OH), compounds I and II formed from peroxidase or heme interaction with H2O2, •NO2 formed from the myeloperoxidase/H2O2/ NO−2 system, hypochlorous acid (HOCl), and reactive species formed from peroxynitrite (ONOO−/ONOOH) decomposition.
Best,
For more information...
doi: 10.1016/j.freeradbiomed.2011.09.030
Dear Venessa,
I don't think DCFDA has to be used with live cells. Base-catalyzed hydrolysis of esters occurs without esterases. I understand you use a buffer, but a small amount of DCFDA likely hydrolyzes to form DCFH which is converted to DCF by H2O2 and other species mentioned by Anderson.
Thanks,
Dear Ran, we use DCF-DA with live renal slices and primary cell cultures and have no problems. Experience of our lab shows DCF-DA as one of best dyes for ROS detecting and quantification in live cells. But instead of Vanessa task we are studying processes, that increase the intrinisic ROS production (ischemia/reperfusion for example).
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