I recently posted a call for suggestions on how to use a
Hoechst/GFP double stain in a cell cycle analysis and I want to correct
an error in my post and address some problems. First, I erred when I
said that we tried shutting off the UV laser and the Hoechst background
is still picked up
in the GFP chanel. Being a fly geneticist and not a flow expert, I
misunderstood the problem. The spillover of the Hoechst into the GFP
chanel goes away when we turn off the UV source. We are trying to
distinguish GFP positive from GFP negative cells at the same time that
we are looking at Hoechst staining, and we have trouble with the
background from the Hoechst obscuring that distinction. The cells do
express GFP from a genomically integrated reporter, but not at a really
high level. So perhaps the problem really is with the cells.
The instrument settings we've been using are as follows: The MoFlo
has the 20 X 60 micron 488nm beam in pinhole #1, and the 20 X 60 micron
UV beam in pinhole #3. Emission path filters are HQ510/20 in FL1 for
GFP and 440/40 in FL8 for Hoescht. 488nm output has consistently been
100mw and we have varied the UV output from 20 to 100mw to obtain less
encroachment.
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