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> Centrifugation after staining with PI and other DNA fluorochromes is not
> necessary for several reasons.
In fact, depending on what you intend to do with the PI signal, it
can be detrimental. Several years ago I performed an experiment in which
I stained nuclei from a human cell line with PI, washed with PBS, and ex-
amined over the course of a few hours. In my control sample (not washed),
the coefficient of the G0/G1 peak was nice and low, around 2%. The G2/M to
G0/G1 fluorescence intensity ratio was very near to 2.00. In the washed
sample, the G2/M : G0/G1 ratio was below 1.90 within a few minutes, and
within a half hour or so the CV of the G0/G1 peak was above 5%. Needless
to say, the intensity also dropped.
Unlike monoclonal antibodies, PI has a fairly high off rate. So
having excess stain in the solution helps to keep all of the binding sites
of the DNA saturated, thus facilitating DNA quantitation. If you just want
to distinguish live cells from dead cells, you can probably get away with
washing away the excess PI. But not for long -- after a few hours the dead
cells would not fluoresce significantly more than the live ones.
> 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.
If for some reason you feel a need to wash cells after staining with
a nucleic acid stain, try ethidium homodimer or maybe the dimeric cyanines.
Unlike ethidium or PI, these have very slow off rates. In parallel to my ex-
periment with PI, I tried ethidium homodimer. Even when washed, ethidium
homodimer-stained cells would give essentially unchanged fluoresence histo-
grams for two or three hours.
> 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