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Wendy,
We have found that ethanol fixation kills the gfp. We have used some of
the non-alcohal fixes such as streck, orthopermeafix but the CV's are not
very nice with pi. You might want to try Hoechst 33342 which does not
require fixation and permeablization for the DNA/surface analysis.
Good luck
Rochelle Diamond
Caltech Flow Cytometry/Cell Sorting Facility
diamond@cco.caltech.edu
On Mon, 24 Feb 1997, Wendy D. Schober wrote:
>
> We are attempting to optimize the procedure to label transfection along
> with cell cycle in HeLa cells. So far we have co-transfected with CD20
> and then labeled the surface with CD20-FITC. We found 5-13% CD20
> positive with good PI patterns after the usual 70% ethanol fixation.
> We used the same cells and fixation procedure with GFP as the reporter and
> hoped to find similar results. Unfortunately, there were few cells GFP
> positive and what may be there are of very low intensity, barely over
> background. We are looking for suggestions for improving our GFP and keeping
> good PI patterns. The GFP is from Invitrogen and they have been somewhat
> helpful, but have not done this with PI.
> Is fixation causing a problem?? If so, is there a better procedure which
> will permeabilize and fix to get both GFP and PI to work?? Ultimately, we
> want this to work in some fibroblast lines, but HeLa is our control.
>
> Thanks in advance for any hints.
> Wendy Schober
> wschober@bcm.tmc.edu
>
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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,
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