Re: your mail

Rochelle A. Diamond (diamond@cco.caltech.edu)
Mon, 8 Jul 1996 10:32:24 -0700 (PDT)

We have been trying to cell cycle studies with GFP in transcient
transfections of mouse cell lines. So far we have
tried a number of fixatives. The alcohal based fixatives wipe out the
GFP fluorescence. Streck fixative was also unhelpful. Orthopermeafix
keeps the fluorescence, but the CV's for the PI are very broad and almost
unreadable, Given that fact, the data that we have gathered so far
indicate that GFP perturbs cell cycle possibly blocking cells in late S
or G2. But as I said the CV's are terrible and we need to do a
cotransfection experiment with a cell surface marker to look at the block.
I would like to try the BD permeablization reagent but haven't gotten it
yet, although I have heard that the CV's with PI are also broad.

I know this doesn't really solve your problem, but at least you know that
you are not alone.

Rochelle Diamond
Caltech Flow Cytometry/Cell Sorting Facility
diamond@cco.caltech.edu

On Tue, 2 Jul 1996, Steven Weintraub wrote:

>
> We are trying to use the enhanced green fluorescent protein (GFP) to
> identify transiently transfected cells for cell cycle studies. We want to
> quantitate DNA in the cells with propidium iodide and assess for new DNA
> sysnthesis with BrDU. Several procedures that we have used to permeabilize
> the cells to allow for PI or BrDU antibody uptake inactivate the GFP. Has
> anyone worked out a method for using GFP in this manner?
>
>
> Steven Weintraub, M.D.
> Departments of Internal Medicine and
> Cell Biology and Physiology,
> Washington University School of Medicine
> at Washington University Medical Center
> Campus Box 8052, 660 S. Euclid Ave.
> St. Louis, MO 63110-1093
> (314) 362-8964 FAX: (314) 362-8987
>
>
>
>
>

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CD-ROM Vol 3 was produced by Monica M. Shively and other staff at the Purdue University Cytometry Laboratories and distributed free of charge as an educational service to the cytometry community. If you have any comments please direct them to Dr. J. Paul Robinson, Professor & Director, PUCL, Purdue University, West Lafayette, IN 47907. Phone: (765)-494-0757; FAX(765) 494-0517; Web http://www.cyto.purdue.edu , EMAIL cdrom3@flowcyt.cyto.purdue.edu

Home Page Table of Contents Sponsors E-Mail Archive Web Sites

CD-ROM Vol 3 was produced by Monica M. Shively and other staff at the Purdue University Cytometry Laboratories and distributed free of charge as an educational service to the cytometry community. If you have any comments please direct them to Dr. J. Paul Robinson, Professor & Director, PUCL, Purdue University, West Lafayette, IN 47907. Phone: (765)-494-0757; FAX(765) 494-0517; Web http://www.cyto.purdue.edu , EMAIL cdrom3@flowcyt.cyto.purdue.edu