Re: More DNA stains...
Howard Shapiro (hms@shapirolab.com)
Sat, 31 Aug 1996 20:57:35 -0400 (EDT)
>1. What if we remove the constraint of excitation at 488 or UV (we will
>soon be getting another laser). Now we are looking for a DNA stain (for
>cell cycle) which can be used on viable cells (to be sorted and
>recultured) which can be excited at any wavelength but which does not
>emit in green or blue.
>
The only dye which has been used reliably by large numbers of people for
stoichiometric staining and sorting of viable cells is Hoechst 33342. A few
other Hoechst dyes (e.g. 33362 and 33378), not available commercially, also
work on viable cells; like 33342, they are UV-excited and emit in the blue
to blue-green, and do not have significant advantages over 33342. Some cell
types can be vitally stained with Hoechst 33258 or DAPI, which are also
UV-excited and emit blue to blue-green. There have been isolated (one
paper, one cell type) reports of stoichiometric DNA staining in viable cells
with mithramycin (blue-violet excitation, green-yellow emission) and
olivomycin (blue-violet excitation, blue-green emission); this has not been
reproducible. The Hoechst dyes, DAPI, mithramycin, and olivomycin are
DNA-specific. Chromomycin A3 and 7-aminoactinomycin D, which are
DNA-specific, have not been reported to stain DNA stoichiometrically in
cells without permeabilization and/or fixation. Occasionally, one can get
near-stoichiometric staining of DNA in viable cells with acridine orange;
the excitation is blue to blue-green and the DNA signal is green. While it
happens, nobody knows how to make it happen reliably.
The cyanine "DNA" stains, including thiazole orange and blue and the TOTO
and TO-PRO dyes and their congeners, as well as the SYTO dyes, all stain RNA
as well as DNA and do not produce a signal indicative of DNA content in live
cells; in general, fixation and/or permeabilization and RNAse treatment are
required to get a DNA histogram using these dyes. Same deal with ethidium,
propidium, and styryl dyes such as LDS 751.
There is probably enough information available to permit rational design of
a visible-excited, DNA specific stain which does not emit in the blue or
green spectral regions; unfortunately, different chunks of it are in the
hands of chemists at different companies which are in commercial
competition. If it were easy to make such a compound, it would almost
certainly be available from Molecular Probes by now.
-Howard
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