Thanks to everyone who has responded to my posts. This has
certainly been educational. To answer a few questions: The Drosophila
cells in question are dissociated imaginal disc cells from dissected eye
and wing discs. I can clearly distinguish GFP negative and positive
cells with a scope. We can also clearly see the difference between GFP
positive and negative cells by flow in the absence of Hoechst staining.
The Hoechst staining is definitely causing the problem with GFP
detection when we try to look at both Hoechst and GFP together.
We have also tried sorting GFP positive and negative cells from
each other followed by separate staining with Hoechst, but many cells
are traumatized by the sorting and die. I've also tried PI, but an EtOH
fix ruins the cells and I'm concerned about using PI on live cells since
that requires permeabilization (which might decrease GFP staining).
Does anyone else have experience with DRAQ5 or another DNA dye
useful for cell cycle analysis on live fly cells? If so, how did you
get it to work?
Does anyone else with experience with fly cells have tips on how to
sort them without traumatizing them? I've heard from other fly groups
that sorting fly cells is rather tricky.
Thanks again.
Renee Read
Department of Molecular Biology and Pharmacology
Washington University School of Medicine
660 S. Euclid Avenue
St. Louis, MO 63110
rdread@artsci.wustl.edu
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