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If you can't change the filters on the FACScan, you've got a big problem.
People have been able to use FITC- and PE- antibodies with PI; to do so, you
need to have a lot of the antigen which will bind the PE-antibody in order
to have any hope of resolving the immunofluorescence in the presence of the
PI signal.
In principle, 7-aminoactinomycin D would be a better DNA stain to use with
FITC- and PE- antibodies; 7-AAD, unlike PI< is DNA-specific, eliminating the
need for RNAse treatment, and its emission is at a much longer wavelength (>
640 nm) than PI's, matching it well to the FACScan's standard filters.
Also, 7-AAD fluorescence is much less bright than PI fluorescence, making
compensation easier. Now, the bad news. 7-AAD is more sensitive than
almost any other DNA stain to chromatin conformation, and has been used by
Stokke, Steen et al to demonstrate differences among cell types in chromatin
conformation, which means that under many circumstances, one doesn't get
good stoichiometric DNA staining. We have found, based on the work of Toba
et al in J Immunol Methods in 1995 and on subsequent suggestions from Dr.
Toba, that staining at a relatively low pH (6.0), before fixation, somewhat
improves CV's with 7-AAD. Jim McSharry, at Albany Medical College, has
stained cells fixed with methanol-acetone with 7-AAD and FITC- and PE-
antibodies to viral antigens; 7-AAD staining is reasonably good and could
probably be improved. However, in work with him, I have come to suspect
that there can be substantial energy transfer between FITC bound to ab's
against nuclear antigens and 7-AAD bound to DNA; this would make it
necessary to compensate between green and red fluorescence, which is, as I
recall, not possible on the FACScan, at least in hardware. On the other
hand, even without compensation, you would probably be able to discern
differences in DNA content distribution among cells bearing one or both
antigens and cells bearing neither.
And by the way, what kind of cells are these?...That and whether you are
looking for hundreds or millions of molecules of antigen will probably be
the major determinants of success or failure.
-Another Howard
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CD-ROM Vol 3 was produced by Monica M. Shively and other staff at the
Purdue University Cytometry Laboratories and distributed free of charge
as an educational service to the cytometry community.
If you have any comments please direct them to
Dr. J. Paul Robinson, Professor & Director,
PUCL, Purdue University, West Lafayette, IN 47907.
Phone: (765)-494-0757;
FAX(765) 494-0517;
Web