Yes, I've had similar experiences. Eukaryotic cell types that endoreduplicate
are sometimes confusing because the line between singlets and doublets, using a
width vs area plot, is blurred as ploidy rises. However, my real difficulties
have been with yeast, where DNA content can reach 16n or more under experimental
conditions. In these cases, the width vs area plot isn't very useful at
all....to me anyway. What software are you using for analysis? We just use
Modfit. I am looking forward to other responses on this subject. I've never
been able to get a straight answer myself.
David McFarland
Howard Hughes Medical Institute
Flow Cytometry Facility
Vanderbilt University Medical Center
---------------------- Forwarded by David McFarland/VUMC/Vanderbilt on 12/17/99
02:21 PM ---------------------------
Derek Davies <daviesd2@icrf.icnet.uk> on 12/16/99 03:26:30 PM
Please respond to derek.davies@icrf.icnet.uk
(Embedded image moved to file: pic16039.pcx)From:(Embedded image moved to
file: pic23159.pcx)Derek Davies <daviesd2@icrf.icnet.uk> on 12/16/99 03:26 PM
To: Cytometry Mailing List
<cytometry@flowcyt.cyto.purdue.edu>
cc: (bcc: David McFarland/VUMC/Vanderbilt)
Subject: A DNA analysis question
Hi all,
This may seem a simple question and one that I feel that I already ought
to know the answer to. When doing DNA analysis by flow it is generally
accepted, is it not, that some form of pulse processing be used to exclude
G1 doublets. On my BD machines I generally use a Width v area scattergram
to define these and exclude. Normally when using nice round cells like
lymphocytes or thymocytes, it is straightforward to see where the G2
population ends and the doublets begin. However, when using larger,
epithelial cells, especially keratinocytes, the distinction is nowhere
near as obvious.
Where we are having problems is in keratinocytes where, for whatever
reason, their mitosis is uncoupled from their DNA replication and they
form 8n cells. My inclination would be to set a gate that runs parallel to
the median of the G1 and G2 populations and extend it upwards to include
potential polyploid cells. The worry is though that there may well be an
increase in pulse width in these cells that further blurs the boundary of
true single cells and clumps. Is there any consensus about how to analyse
these sorts of plots? I generally now collect samples ungated and play
around during the analysis stages. However I think that it may be
difficult to estimate the true polyploid cells. I have tried analysing
some of these samples on a Laser Scanning Cytometer but the distinction
between clumps and polyploid and/or multinucleate cells is also not clear.
Anyone have similar problems or any ideas?
Derek
************************************************************************
Derek Davies Voice: (44) 0171 269 3394
FACS Laboratory, FAX: (44) 0171 269 3100
Imperial Cancer Research Fund, e_mail: derek.davies@icrf.icnet.uk
London, UK
Web Page: http://www.icnet.uk/axp/facs/davies/index.html
In tenebris lux
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