Re: High speed sorting
SEK-Claude Cantin (cantinc@IRCM.UMontreal.CA)
Wed, 5 Apr 1995 09:37:05 -0400 (EDT)
Just thought this might help for those who haven't already tried:
Increasing cell concentration up
to 50 million cells per ml for "high speed" sorting on an "standard"
Facstar Plus allows increased throughput rates while actually
improving purity. The theory is that sample differential pressure can be
reduced significantly (in fact, I get no reading on the LED's) and still
obtain 'high' cell throughput rates; the effect is that the
sample core is narrowed at low differential pressures, and therefore cells
pass through the nozzle in single file (more or less) instead of side by
side (sort of) which is more likely with a large sample core diameter
(i.e. higher sample differential pressures). The number of coincidences
is diminished at high cell concentrations and low sample differential
pressures, therefore improving purity and recovery.
I routinely do my sorts at high concentrations, cells running at 4,000 to
5,000 per second, and consistently obtain 99.9% purity; controlling the
flow rate is a little more difficult however, and a recent sample sorted
a few minutes at 8,000 cells per second before I caught it...still, purity
was 98.5% from a starting population which looked like a double camel hump
and was 21% positive at the start...and some of that is due to pos/neg
doublets getting through. I have no numbers for recovery, but
what I observe is no significant loss (except at those rates you use up cells
pretty fast during setup). High cell concentrations may cause other
problems (clumping / clogging), but if you can get around that, 5,000
cell per second sorts on a Factsar Plus is no sweat. I haven't yet tried
to establish an upper limit though. Thanks to a couple of guys from B.D.
for the idea.
CLAUDE CANTIN
cantinc@ircm.umontreal.ca
CYTOMETRIE EN FLUX / FLOW CYTOMETRY
INSTITUT DE RECHERCHES CLINIQUES DE MONTREAL
TEL. 514-987-5608
FAX 514-987-5633
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EMAIL robinson@flowcyt.cyto.purdue.edu
