Dear Renee,
I saw the similar phenomena when I use HO/GFP or DAPI/FITC double staining
in cell cycle analysis on MoFlo. Especially when UV was set on 2nd pinhole.
But, there was a way to fix the spillover problem by using software(Summit
3.1) compensation. I used following steps during acquisition and analysis.
1) Acquire GFP or FITC single positive tube and save the file as usual.
2) Set up voltage for HO or DAPI channel by choosing Lin and Int on
electronic rack. You can use double or single stained tube.
3) Select log and peak for HO or DAPI channel on electronic rack.
Acquire the same tube for step 2). Don't change the voltage. Compensate
between HO and Fl1 channels by using compensation Matrix and save the file.
4) Change back to Lin and Int for HO or DAPI channel on electronic rack.
5) Run your double stained sample and save the file for re-analysis
(save both uncompensated and compensated data).
6) Re-analysis. Attached file showed you the layout of the re-analysis.
Compensate by using the file saved on step 3) and then pull the file saved
on step 5) to analysis. The plot of HO Comp vs. GFP comp(in the
middle-top)was the right plot to look at. The percentage of GFP+ was very
similar to that from single positive tube(file saved in step 1). The plot of
DSP(comp HO) vs. DSP(comp GFP)(on right) was exactly what you saw during
acquisition(step 5).
If the cells have weak expression of GFP or FITC+, there is no way to get
the correct result without fixing the spillover problem.
I hope it helps.
Hongmei Shen Ph.D.
Associate Director
Flow Cytometry Core Facility
University of Pittsburgh, Cancer Institute
200 Lothorop St.
W 946 Biomedical Science Tower
Pittsburgh, PA 15213
Tel. 412-648 7217
Fax. 412-624 9624
e-mail: shenh@msx.upmc.edu
-----Original Message-----
From: Renee Read [mailto:rdread@artsci.wustl.edu]
Sent: Friday, April 19, 2002 6:08 PM
To: cyto-inbox
Subject: Hoechst, GFP, and cell cycle post error
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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