The best way is to analyze live cells that are not fixed, in the presence of 1 ug/ml propidium iodide to exclude dead cells. Dont use a hard gate to exclude PI+ cells - instead look at FL3 vs FL plots to gate on GFP+, PI-negative. ======================================================= Rachel M. Gerstein, Ph.D. Department of Molecular Genetics and Microbiology Graduate Program in Immunology/Virology University of Massachusetts Medical School 55 Lake Avenue North Worcester, MA 01655-0002 (508) 856-1044 (508) 856-5920 (FAX) > ---------- > From: Nicholas Sibinga > Sent: Tuesday, October 12, 1999 12:57 PM > To: Cytometry Mailing List > Subject: gfp and facs analysis > > > Hello -- > > I'm hoping that you can give me some advice or protocol information on > FACS analysis of transiently-transfected cells expressing green > fluorescent protein. I am planning to use a co-transfected EGFP plasmid to > serve as a marker for transfection, but I am concerned that fixation > methods (such as those used for propidium iodide analysis of DNA content) > may decrease the GFP signal. Can you advise the best way to do this? > > Thanks very much, > > Nicholas Sibinga, MD > > Cardiovascular Biology Lab > Harvard School of Public Health > 677 Huntington Avenue > Boston, MA 02115 > > phone 617-432-3586 > fax 617-432-2980 > >
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