> >I have a client who wants to do cell cylce on GFP transfected cells. Since >I do not have a sorter, I need to be able to do this on a Calibur. She has >found some comments that claim you can do DNA with a formaldehyde fix >rather than ethanol. Is that true? Also, I read on this list a while back >that PI may be so bright that it may overwhelm low GFP signal so 7AAD would >be the better choice. I have not been able to find a protocol for cell >cycle with 7AAD. Any help would be greatly appreciated, as always. > >Thanks, >Candace Enockson >Medical University of South Carolina Greetings candace, Fix your cells in 1% formaldehyde overnight at 4'C prior to staining with 50ug/ml PI, 500ug/ml RNAase if you can leave the cells longer in the fixative (>2days the DNA profiles improve somewhat) I have used this method to get cell cycle/GFP data on herpes transduced CD34+ cells (see Gene Ther 1998 May;5(5):718-22) I don't recall there being any problems of signal crossover between GFP/PI that could not be fixed by dialling in modest amounts of compensation. regards, Arnold _/_/_/_/_/_/_/_/_/_/_/_/_/_/_/_/_/_/_/_/_/_/_/_/_/_/_/_/_/_/_/_/_/_/_/_/ Arnold Richard Pizzey Department of Haematology Royal Free and University College London Medical School 98 Chenies Mews London WC1E 6HX U.K voice: +44 020-7679-6234 Fax: +44 020-7679-6222 email: a.pizzey@ucl.ac.uk _/_/_/_/_/_/_/_/_/_/_/_/_/_/_/_/_/_/_/_/_/_/_/_/_/_/_/_/_/_/_/_/_/_/_/_/
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