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First, was the PI added after fixation to assess DNA content or before to
assess membrane integrity?
Second, are you following the manufacturer's fixation
method-paraformaldehyde followed by ethanol or some different method?
In our hands, cells fixed in para do not exhibit any subdiploid peak at all.
You may want to verify this for your system, but if this is true, perhaps a
trigger on PI fluor. could be brought right up to the G0 peak. The
assumption is anything below this level is not an intact cell.
For the regular subdiploid method, I trigger on 25% of the DNA content of
the viable cells. You can change the position of the A0 peak by time after
fixation or the buffer you store the cells in. The theory is you are
looking for cells with a slightly reduced DNA content and want to exclude
mitochondria, apoptotic bodies, etc.
Also, the para/EtOH fixation step tends to cause a lot of cell loss
resulting in debris and a very messy LS hist. Depending on the stimulus you
are using to induce, this effect could be exacerbated. We have found that
the Ortho reagent Permeafix gives a much cleaner LS histogram with reduced
cell loss.
You don't mention which cell type you are working with, but it may be
possible that a LS trigger would be more appropriate. The way you feel most
confident that you are looking at whole, intact single cells is the way to
go.
Good luck.
-------------------------------------
Name: Tom Mc Closkey
E-mail: thomasm@nshs.edu
Date: 08/08/96
Time: 17:15:11
This message was sent by Chameleon
-------------------------------------
Thomas W. Mc Closkey, Ph. D.
Director, Flow Cytometry
North Shore University Hospital
Cornell University Medical College
Manhasset, Long Island, New York
ph: 516-562-4641 fax: 516-562-2866
e-mail: thomasm@nshs.edu
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