[Cytometry] Cell cycle profiling during long time course -non-lytic fixation

W.E.Corver@lumc.nl W.E.Corver at lumc.nl
Fri Feb 19 05:53:01 EST 2010


I never worked with CellFix, but it might contain a detergent. Our experiences with formalin is that it doesn't lyse cells and a short fixation on ice doesn't perm the cells. In the past we performed many experiments with cultured tumor cells and with dissociated cervical and breast tumors using 1% PF (formalin) and lysolecithin or 1% PF in combination with methanol. We used PI as indicator for permeabilization, but also for DNA ploidy after permeabilization. By titration you can nicely observe the affect of the permeating agence on permeabilization. We published on several occasions using these method (see Cytometry and other Journals, type my name in PubMed). However lysolecithin does lyse red blood cells even after formalin.

Also many other groups used formalin in combination with methanol. Formalin / 50% methanol is excellent for phosphor-flow (Jacobberger, Shankey, Hedley)  and FSC/SSC characteristics are maintained.

 

Regards,

 

Willem Corver

________________________________

Van: cytometry-bounces at lists.purdue.edu namens Robin Barclay
Verzonden: vr 2/19/2010 11:02
Aan: cytometry at lists.purdue.edu
Onderwerp: Re: [Cytometry] Cell cycle profiling during long time course -non-lytic fixation



The reply below has prompted me to pose the following question to the flow
community. We have been in the habit of using commercial fixative (BD's Cell
Fix) following staining to give us the option to postpone running samples
for flow analysis until the next day (or even next again day).  There are
some minor shifts in FSC/SSC (compared to unfixed cells), but generally if
we always run stained samples in CellFix then that becomes constant.
Recently we have been interested in early (and not so early) red cell
progenitors found naturally or arising in various HPC cultures after various
incubation times, and note that as they mature they become increasingly
lysis sensitive.  BD's CellFix is very lytic - which we had not appreciated
because we had run many samples for other investigations as whole blood
stains followed by lyse/wash before suspension in CellFix. Can anyone
recommend a fixative for stained cells which will not lyse red cells -
immature or mature?
Robin Barclay

Dr George Robin Barclay PhD
Consultant Clinical Scientist (Lead Scientist, SNBTS Group)
  & Honorary Senior Lecturer
SNBTS Cell Therapy R&D Group
MRC Centre for Regenerative Medicine
The Chancellor's Building
University of Edinburgh
49 Little France Crescent
Edinburgh EH16 4SB

Tel: 0131 242 6259 (26259) office/ 242 6266 (26266) lab
e-mail: robin.barclay at ed.ac.uk


--
The University of Edinburgh is a charitable body, registered in
Scotland, with registration number SC005336.


----- Original Message -----
From: <W.E.Corver at lumc.nl>
To: <ag586 at cam.ac.uk>; <cytometry at lists.purdue.edu>
Sent: Thursday, February 18, 2010 11:05 PM
Subject: Re: [Cytometry] Cell cycle profiling during long time course


> Dear Antonis,
>
> Formaldehyde (formalin, because formaldehyde is a gas and we had this
> discussion before on this list) can be used without any problem. Keep the
> fixation short and on ice. 1% to 4% will do. The longer you fix and the
> higher the concentration of formalin you use, the higher your CV of the
> G1. Next wash and perm with ethanol or methanol. You can find numerous
> papers in Cytometry or Current Protocols using these methods. Keep the
> cells swirling while adding the fixatives drop-wise to prevent clumping.
> You can also use the Vindelov method (Cytometry 1983), which indeed is
> based on lysing! You can store the nuclei suspension for several days in
> the frige. Results are excellent!
>
> Regards,
>
> Willem Corver
> LUMC
> Netherlands
> ________________________________
>
> Van: cytometry-bounces at lists.purdue.edu namens Antonis Giakountis
> Verzonden: do 2/18/2010 16:59
> Aan: cytometry at lists.purdue.edu
> Onderwerp: [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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