Using DHE to measure ROS: please help! - When should I incubate DHE? (May/12/2011 )
Please discuss this with me.
I am using dihydroethidium (DHE) as a probe to detect ROS production after drug treatment. In the majority of the protocols I saw that the authors incubate DHE in the last 30 minutes of treatment (for instance, if I want a 10h treatment with the drug I would incubate DHE at the 9h30m time point). Then, it's just washing and reading in the cytometer. However, in other (not so common) procedures I saw that the authors incubate DHE in the beggining of the experiment: they incubate the cells with DHE (30 minutes), wash, and then they do the drug treatment.
Which of the options is the correct one? I mean, concerning the second option, is DHE recycled so I can detect ROS production throughout the whole time course of the treatment? And what happens to the DHE that is oxidized in the first minutes/hours of treatment? Is it kept inside cells or lost to the extracellular medium?
Concerning the first option, 30 minutes of incubation in the end of the treatment is reliable enough to have a global impression of ROS production that is occuring?
Right now I am completely puzzled, because apparently I have different results when comparing the two methods...
If you are trying to develop the method, then this would be a nice topic and you can dig into it.
If you just want the result, I suggest you purchase a kit and do exactly the introduction said.
I might confuse you more but when I used DHE for the same reasons, I incubated my cells with it for 20' before I applied my drugs. However it worked pretty nice. You have to keep in mind that DHE must be applied in Ar atmosphere to avoid oxidization by air. Although too late, I hope it was a bit of help, if you 're still doing these experiments.