I've been trying to adjust a protocol for ROS staining using DCFDA and am unsure of what to use for a single color control for compensating the cytometer. I've tried using stimulated cells stained with DCFDA to get a positive population, however that hasn't worked as well.
I've resorted to using CD11c FITC antibodies to mark a positive signal with the dendritic cells but am not sure if that is the best approach as results have been mediocre at best.
Have you try to use H2O2 as a positive control and N-Acetyl-Cystein as a negative control?
It depends on what you want to do and why do you need the compensation. Do you really need to do a multicolor panel? If you want to get MFIs for quantification there's a lot to be said for single channel designs.
It is indeed probably best to create a positive control by stimulating with a ROS generating agent. You can do direct ROS with H2O2, which should work fine or you can use something like menadione that will generate ROS indirectly. If for some reason that doesn't work you could indeed use a FITC labelled antibody. The spectral profile should be very similar as DCFDA is a fluoresceine based compound.
Lastly you might want to consider a different way of measuring ROS. CM H2 DCFDA might work better than regular DCFDA for FCM as it doesn't leak out of the cells as quickly (in theory). Since you appear to be investigating DCs (judging from the CD11c) you might also try Rhodamine 123, if phagocytosis is part of your question.
I agree with Eros & Hendrik, proper negative and positive controls might be necessary for your case. If feasible, use CD11c - PE / Rhodamine conjugated.
In the past for performing uptake-evaluation studies, we adopted a dual-staining strategy for DCs, wherein CD11c FITC and Rhodamine were used (please see Fig. 2 for details). Therefore, combination of CMH2DCFDA and Rhodamine might prove to be a better strategy, as suggested by Hendrik.
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I think that the combination of this three answer will help you to adjust your protocol! It is very important to use H2O2 and NAC as control as ROS are not very stable!! I tried to detect ROS in macrophages RAW264.7 with cell ROX and it works also.... but with a simple staining
https://tools.lifetechnologies.com/content/sfs/manuals/mp10422.pdf
Best!!
All the answers above are very good and should help you with the anlysis. However, it seems that you did not dissolve the compound properly. The compound precipitates easily thus you have to make sure it is completely dissolved in ethanol you have to mix well the compound. As the previous collegues suggested, the mixture can be analyzed easily by adding peroxide and see thee changes in emision. So before adding to the cells use a fluorimeter and assess the changes in emission just by adding DCFH to peroxide, it should turn yellow, if it does not then it is simply not dissolved. Good luck.
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