I want to do a sorting experiment by staining my live mouse ES cells with Hoechst 34580, sort them in different cell cycle phases and fix and stain each sorted cell cycle phase population with another DNA-binding dye to check if their DNA-profiles match to the gates we sorted on. My question is if I can use a dye like PI, which binds the DNA in an intercalating way, after I stained with Hoechst which binds the minor groove.Are the dyes going to interfere? Does anyone have experience with this?
You can use 7-Aminoactinomycin D (7-AAD). This is very specific dye and can give you exact phase of your cell cycle. I have personally used it in a multi-color flowcytometry experiments.
Good luck!!
You could also use methyl green. It binds to the major groove, fluoresces in the far-red, and gives you the chance of using specific antibody staining in the green-red portion of the spectrum at the time.
I would think many intercalation agents would bind readily in the presence of Hoechst compounds. For a reference see J Phys Chem B. 2007 May 17;111(19):5047-52. Epub 2007 Apr 25.
Simultaneous binding of minor groove binder and intercalator to dodecamer DNA: importance of relative orientation of donor and acceptor in FRET. This study examined Hoechst binding in the presence of ethidium bromide.
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