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You may also consider a more aggressive physical disruption of your cells by
putting them on a sheet of nylon mesh, folding it over on top of the cells
and then running a robber policeman or spatula over the top. I have used
this physical disruption, coupled with DNAse to make single cell suspensions
from tissue.
Calman Prussin
------------------------ Forwarded Message -----------------------
Date: 6-18-1995 3:50pm
From: {raymond.chang@icf.med.uni-muenchen.de}:unix:niaid
To: calman prussin:10:niaid
Subj: How to get a monocellular cell suspension? (fwd)
Also-to: cytometry mailing list <cytometry@flowcyt.cyto.purdue.edu>
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Mariangela posted the question of how to get good cell suspension.
Normally, cancer cells is much easier than primary cells to get
monocellular suspension. You should use small volume to resuspend the
cells by pipetting up and down several times, then adjust the volume to
what you need. If there is still a portion of big clamp, for our experience,
we will discard these clamps by filtering the cells through a 100micron
nylon mesh. If your cells are small, you can use the nylon mesh in smaller
size. Through these procedures, we get a very good monocellular
suspension. Hope this information is helpful.
Raymond, Cheun-Chung CHANG
DAAD Research Fellow,
Institute for Surgical Research,
Klinikum Grosshadern,
University of Munich,
D-81366 Munich.
Germany
Fax: +49-89-7095-8897
---------- Forwarded message ----------
Hi! I am inducing differentiation in an anaplastic carcinoma cell line
using tyrosine kinase inhibitors in vitro. After treatment the cells
differentiate and form desmosomes. I harvest them for cell cycle analysis
and sorting, using trypsine (0.05%) with EDTA (up to 5 mM), but I still do
not obtain a monocellular cell suspension from my culture. After
trypsinization some of the cells are in suspension but some of them form
big clumps and the desmosomes are still there! Does anybody has any
suggestion about that?
Thanks!
Mariangela Mancini
e-mail mmancini@fred.fhcrc.org
Fred Hutchinson Cancer Research Center
Seatlle,WA
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