[Cytometry] Cell cycle profiling during long time course

Sharma, Parul Parul.Sharma at childrens.harvard.edu
Thu Feb 18 11:42:45 EST 2010


Hi Antonis,
In cell cycle analysis we usually measure the number of dividing (proliferation index) cells. These cells will have two nuclei i.e in the M phase.
Whereas the cells in Go/G1phase will have one nuclei per cell. In S phase the amount of DNA is intermediate between these two phases.
Hope this helps
Parul

-----Original Message-----
From: Antonis Giakountis [mailto:ag586 at cam.ac.uk]
Sent: Thursday, February 18, 2010 11:26 AM
To: Sharma, Parul
Subject: Re: [Cytometry] Cell cycle profiling during long time course


Hi Paul,

Thanks for your reply. I am not sure I understand your e-mail. I lyse the
cells and then sort nuclei, measuring DNA content. There is a single nucleus
per cell with different DNA contents. Before incubating with Hoechst and
prior to cell lysis, I count the cells ensuring that I have equal densities
among my samples.

Antonis
----- Original Message -----
From: "Sharma, Parul" <Parul.Sharma at childrens.harvard.edu>
To: "'Antonis Giakountis'" <ag586 at cam.ac.uk>; <cytometry at lists.purdue.edu>
Sent: Thursday, February 18, 2010 4:17 PM
Subject: RE: [Cytometry] Cell cycle profiling during long time course


Hi Antonis,
If you lyse the cells how are you going to do cell cycle analysis? You will
have no way of gauging the number of nuclei within a cell?
You can preserve the nuclei in ice cold methanol or acetone if you believe
the cell membrane is still intact after the cell lysis procedure. Parul



-----Original Message-----
From: cytometry-bounces at lists.purdue.edu
[mailto:cytometry-bounces at lists.purdue.edu] On Behalf Of Antonis Giakountis
Sent: Thursday, February 18, 2010 11:00 AM
To: cytometry at lists.purdue.edu
Subject: [Cytometry] Cell cycle profiling during long time course


Dear all,

I would like to profile cell cycle with flow cyt during a long time course.
The experiment will last 8 h or more. When I do standard cell cycle
analysis, I lyse the cells, incubate with hoechst and then analyse. My
problem is that by the time that I collect the samples from the time course
till the time I analyze them there will be many hours, possible an O/N. I
therefore must find a way to preserve the samples before the analysis,
without changing the cell cycle profile. I know that formaldehyde fixation
is not good and I do not ethanol fix my cells.

Any suggestions? Many thanks for your time,

Antonis

--------------------------------------------
Antonis Giakountis, PhD
Prof. David Baulcombe group
Department of Plant Sciences
University of Cambridge
Downing Street
Cambridge CB2 3EA
UK
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