Cell clumps.

Newsom, Brian S. bsnewsom at msmail.his.tch.tmc.edu
Fri Jun 26 11:14:00 EST 1998


At least part of the problem is almost certainly biological. We have had   
similar problems with skin fibroblasts and other large adherent lines.   
The best thing is to go ahead and have them resuspended in media instead   
of PBS. The phenol red may cause minor problems but nothing compared to   
the problems the clumping can cause. The other thing we do is that   
instead of filtering these cells we let them sit for about 5 min. and   
then pull off what has not settled, we seem to get better recovery than   
using the filters and what is left in suspension doesn't seem to clump as   
bad. There maybe something biologically different about these cells that   
causes them to settle that may be interesting, but that's another problem   
in itself. Like you said keep everything cold, if you don't have a   
chiller for the sample block you may want to look into it, they help a   
great deal for some cell types. The one thing that sounds funny is the   
coincidence rate, it seems to high for a MoFlo at that speed. You may   
want to post that part on the MUG list and see what others have to say   
about it.

Brian Newsom
Center for Cell and Gene Therapy
Baylor College of Medicine

From:  Bill Nostrom
Sent:  Friday, June 26, 1998 9:01 AM
To: cyto-inbox
Subject:  Cell clumps.

Hi  All.

The other day, we had a user who wanted to sort an adherent lung
carcinoma cell line (H157). He trypsanized the cell according to
procedure, and brought them over in PBS without Calcium or Magnesium. We
filtered the cells trough a 30 micron mesh filter and ran on our MoFlo,
at a DDF of 92K, and voltage of 15.5. We ran  cells at 60 psi, but
because of the low concentration (we reconcentrated them ,but it was
still less than optimal) the flow rate was only about 1,000 cells/sec.
The coincidence rate was 30%, and the sort rate was only 10%. We were
trying to sort out the top 25% of the entire population. There should be
no way that those numbers could be correct. After we sorted, we took a
look at the presorted cells under a microscope, and noticed that they
had started to clump back together. So, with all of that information,
can anyone give me an idea of how to keep these cells in a single cell
suspension?  Of course we kept everything cold, and didn't do anything
different than any other sort. this is the first time, however, that we
tried to sort this particular cell line. Is there some biological
explanation? Any help is appreciated. Thanks,
Bill Nostrom
University of N.C.

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