Johana, Inspection by microscopy following your dissociation treatment should give you an idea of the effectiveness of your nuclei isolation. Re. identifying these nuclei in flow . . . think of a DNA counterstain. Given that your technique leaves the chromatin intact, you should be able to find DNA content signals similar to that of permeabilized whole cells. Trigger on the DNA stain's signal, then look for these events in your light scatter, or, go straight to evaluating your protein. MAK. -- Mark A. KuKuruga, Managing Director University of Michigan Flow Core 7416 CCGC 0946 (734) 647-3216, fax (734) 936-7376 kukuru@umich.edu >>> "Melendez, Johana" <MelendJ@Moffitt.usf.edu> 12/22/01 05:55 AM >>> Hello everyone! I'm new at this list and so far I've learn many things from you. Now I need your help on how to set FSC and SSC to view intact nuclei (that has been separated from the cell). I just want to make sure that I gate on real nuclei, not on debris or whole cells that may may appear in the preparation. It is necessary to gate out anything that is not nuclei only, since our interest it is to study a translocated protein that should appear inside the nuclei upon stimulation. If we don't identify the real nuclei, we could be study the protein that is outside the nuclei, that has not been translocated (and that it is not of our interest). Any suggestions will be appreciated. Johana Melendez H. Lee Moffitt Cancer center Tampa, Fl.
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