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1) PI bound to DNA has a quantum efficiency at least 50 times that of PI
free in solution.
2) The concentration of PI in stained nuclei is much higher than that in
solution.
3) The flow cytometer is effectively looking at a volume of a few thousand
femtoliters or less, so it won't pick up that much background fluorescence.
4) The electronics in the PMT preamplifier remove the DC background
contribution from the fluorescence signal.
Reason 1 applies to PI, ethidium, the chromomycin dyes, 7-aminoactinomycin
D, DAPI, the Hoechst dyes, styryl dyes such as LDS751, and the monomeric and
dimeric cyanines of the TO-PRO, TOTO, and SYTO series. Other dyes, such as
oxazines and acridine orange, do not increase quantum efficiency markedly on
binding to nucleic acid and may actually be quenched somewhat; background
fluorescence with these is higher but reasons 2-4 still work. Reasons 2-4
also explain why one can do immunofluorescence without washing using
directly labeled monoclonal antibodies.
-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