Hi Naila

It's hard to say. But your DHE is dihydroergotamin? Why I ask, because it is also a drug. 

So if I understand good your assay model contain 2 different drugs, and you want to know if you should wash first one before adding second one? If I were you I would like to check all 3 possibilities of this model, and than deicde is I should wash it or not.

Enjoy your experiment :)

Mike

Hello Naila,

Yes, washing is very important....As I can get, DHE you want to do for FACS...so you need to wash the cells as that will result in washing out of all residual drug and DHE can bind efficiently without much background signal.

If its a drug as per Michal then also washing is important because any residual drug can interfere with activity of your second drug. If you want to observe synergistic effect then you can put both drugs in equal concentration. There is no point of incubating other drug when second drug is still in system in some residual amounts of which you have no ides (how much it is)...so best is, to go with washing step first and then giving incubation of DHE.

Hope it helps !

By DHE do you mean dihydroethidium?

If so then it might depend on your drug. When I have used DCF in the past I just added directly to the culture medium for the final 15 - 30 min of the drug treatment. It is probably more important to wash your cells prior to analysis. Even then, since DHE and DCF are reactive dyes you would be washing away unreacted and non-fluorescent dye so it should not affect your result.

thanks yup i want to check the effect of one compound on hydrogen peroxide production but i wonder do i need to wash the drug after treating cells before adding DHE as it won't affect the levels of ROS in the system. if yes then do i need to wash with PBS? or just the media?