Ian, I don't think you will have any problem unless your particular cells have an ungodly amount of MDR or MDR like protein overexpression. We have looked at a number of reagents on sp cells and to date I have not noticed anything like this that leaks so badly as to totally exclude the reagent. The difference in Hoechst fluorescence is a relative thing. The normal sp (meaning cells from BM, PB thymus and the like) cell excludes more dye than the non sp cells but they are by no means pumping out all of the dye and therefore it should be a similar phenomenon with FDG. If you are using a cell line that has high P-glycoprotein expression it may be an issue but it should not be using primary human or murine cells. Brian Newsom Director, Flow Cytometry Center for Cell and Gene Therapy Baylor College of Medicine -----Original Message----- From: ian titley [mailto:iant@icr.ac.uk] Sent: Friday, December 17, 1999 5:52 AM To: cyto-inbox Subject: FDG and MDR Dear All We plan to look, by flow cytometry, at gene expression in cells of the "side population" as described by Margaret Goodell and colleagues. These are cells which show "dim" Hoechst 33342 staining when viewed on a bivariate blue/red plot. My understanding is that they have a "multidrug resistance" phenotype and are distinguishable because they pump the 33342 dye out. We intend to look at gene expression via the lacZ reporter and FDG. Again my undersatnding is that the fluorescein product of the enzyme reaction "leaks out" of the cells with time and my worry is that in these MDR type cells this process will be enhanced and we may see no signal. We can not use verapamil as this removes the side population from view. My question is: does anyone have experience of looking at FDG in MDR cells and does it work? and if not can anyone think of a more stable (? anchored) product which we could use? (we are stuck with the lacZ system). All the best for the festive season Ian Ian Titley PhD Leukaemia Research Fund Centre at the Institute of Cancer Research 237 Fulham Road LONDON SW3 6JB UK Tel Direct: +44 (0)207 970 6048 or Tel: +44 (0)207 352 8133 ext5134 Fax: +44 (0)207 352 3299 E-mail: iant@icr.ac.uk
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