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@msn.com or dcoder1@hotmail.com tel./messages: 206-499-3446 ----- Original Message ----- From: "Ryan Duggan" <rcduggan@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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