Re: DNA staining
From: Larry Arnold (lwarma@med.unc.edu)
Date: Tue Dec 19 2000 - 16:19:01 EST
Mario
Many of the people using our facility seem to like to use 5-20ug/ml of
PI. At this concentration we see substantial movement of the G1
(and G2/M) peak position and can correlate this with the cell
concentration which they often vary considerably(+/- a couple-several
million/ml). I think you are right that at some concentration the
PI has to be saturating over a fairly wide cell concentration but I don't
know what that is but may be around the usually recommended concentration
of 50ug/ml. I think the main point is that to get good data (ie.
CVs) one has to make sure equilibrium is reached and maintained
(don't dilute the cells in something that doesn't contain the same [PI]
).
If you want to avoid needing to move software peak finders around a lot
or want to overlay histograms (does FlowJo have the ability to shift
(transform) data along a linear parameter?) then you need to try to
maintain as close a constant cell:PI ratio or use a higher concentration
of PI. Higher concentrations of PI can however cause problems with
FITC signal in some cases - the main reason some applications require the
lower concentrations I noted. Maybe somebody should do (if it
hasn't been done - I couldn't find it in a quick search) a careful
experiment to measure this. Perhaps somebody also knows the PI
binding capacity of DNA such that we could determine how close we are
operating at different PI:DNA concentration ratios.
If we accept that differing PI:cell ratios changes the PI
binding/fluorescence and it is not due to changes in the concentration of
PI:DNA then what else could it be? It comes to mind that it
is known that ion concentration changes the PI fluorescence - low ionic
strength buffers give better fluorescence than isotonic buffers.
Could the cell concentration change this? Don't see it but maybe
but it is testable. Perhaps some other effect of higher cell
concentration shifts the PI binding or fluorescence quantum
efficiency.
Let me know if I'm not making sense.
Larry
At 02:56 PM 12/19/2000 -0500, you wrote:
Larry, I agree that it's an
equilibrium situation, and that time, temperature, and PI concentration
are all important factors. However, it seems to me that cell number ought
to be nearly irrelevant (at least within certain bounds). The
concentration (amt) of PI available is so hugely in excess of the final
amount bound at equilibrium, that the cell concentration should have
absolutely no effect on final fluorescence. Of course, if the
amount of PI bound becomes more than a few percent of that in solution,
then this isn't true anymore. But I find it hard to believe that
this is the case...
It seems that you should be able to stain as much as a few hundred
million cells without running into this problem. Am I wrong?
mr
(PS, I didn't post this to the mailing list, but feel free to post it w/
a response if you wish)
Larry W. Arnold, Ph.D.
Res. Assoc. Prof.
Director, Flow Cytometry Facility
Department of Microbiology and Immunology
Lineberger Comprehensive Cancer Center
CB# 7290
University of North Carolina at Chapel Hill
Chapel Hill, NC 27599
Phone: 919-966-1530
FAX: 919-962-8103
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: Wed Apr 03 2002 - 11:56:17 EST