DNA comparison

David Coder d_coder at MSN.com
Thu Mar 7 12:37:12 EST 2002


The simplest first thing to try is to trigger on PI fluorescence--you can
exclude all particles lacking the fluorescence intensity of cells or nuclei.
Adjust the threshold such that you include G0,1 cells. You should be able to
see cells/nuclei among the debris. Also, a pulse area measurement of PI
fluorescence will give the more accurate stoichiometry of PI fluorescence to
DNA content. (And you are using RNase to get rid of PI-labeled double
stranded RNA?).

Dave
========================================
David M. Coder, Ph.D.,
Consultant in Cytometry
email: d_coder at msn.com or dcoder1 at hotmail.com
tel./messages: 206-499-3446
----- Original Message -----
From: "Ryan Duggan" <rcduggan at midway.uchicago.edu>
To: cyto-inbox
Sent: Tuesday, March 05, 2002 2:56 PM
Subject: DNA comparison


>
>
> We're trying to estimate the amount of DNA in the cells of a crustacean
> species by comparing the PI fluorescence of it to PI fluorescence of
> Drosophila cells.  We are having problems both with the sample prep of
> drosophila (embryos or whole flies) and crustacean (we basically grind
them
> up) and staining of the cells.  Our major problem lies in the fact that we
> get so much debris that it is difficult to find populations of cells (or
> nuclei).  If anyone has done anything similar to this, your experiences
> would be helpful and if anyone has specific experience measuring DNA in
> drosophila or crustacean-type species, that would be greatly appreciated
> also.  We are using a BD LSR to analyze; 20ug/mL PI staining after
> detergent permeabilization (not fixed), and varying concentrations of
cells
> (1x10^5 cells to 5x10^6 cells)
>
>
> Thank You,
>
> Ryan
>
>
>





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