Re: DNA + surface staining
Howard Shapiro (hms@shapirolab.com)
Thu, 9 Jan 1997 22:27:29 -0500 (EST)
>I'm looking for a realiable method of staining bone marrow cells for
>simultaneous cell cycle analysis and 3-color surface
>immunofluorescence. Antibodies are FITC-, PE-, and biotin-conjugated.
>I'm using a FACStarPlus with Argon, He-Ne, and UV lasers. Can anyone
>suggest a good DNA stain and avidin conjugate that will work for this
>configuration ?
>
I'd definitely use Hoechst 33342 as the DNA stain. I haven't personally
used APC-avidin (my APC experience has been with direct conjugates), but APC
would be the best choice of label for your third immunofluorescence color
since you shouldn't need compensation. The reason the Hoechst/FITC/PE/APC
combination isn't very widely used is that relatively few of us have the
luxury of 3-wavelength excitation.
You haven't mentioned whether you want to do this with live or fixed cells;
with live cells, you may need a pharmocologic fix to get the CD34+
population to stain well with the Hoechst dye. With fixed cells, you can
stain with Hoechst (1 ug/ml) after the cells are stained with antibody and
fixed; CV's are improved by addition of 0.05%-0.1% Triton X-100. Live cells
generally require higher Hoechst dye concentrations.
-Howard
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