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I have a user who is transfecting cells with a construct for a cytosolic
GFP. We are then assessing the cell cycle status of the GFP+ve cells using
Hoechst 33342 as the DNA stain. Thats fine. NO problems.
However, due to time constraints, accessibility to the UV laser and the
large number of potential samples, what we also want to be able to do is
perform the same assay on fixed cells. Ethanol fixation abolishes or
greatly reduces GFP staining, and paraformaldehyde fixation leads to
insufficiently peaky DNA histograms. Now before we re-invent the wheel, is
there anyone who can point us to the best way of doing this. Would PF then
post-fixing in ethanol be better? Or detergent? A different GFP? Any
suggestion gratefully received!
TIA,
Derek
Derek Davies
FACS Lab
Imperial Cancer Research Fund,
London, UK
http://www.icnet.uk/axp/facs/davies/index.html
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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