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>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;
Here's a hint: There are two POTs behind the diff. pressure display that
you can turn to so you can see your LEDs at even at zero diff. pressure.
>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.
That theory doesn't hold water considering the amount of dead time in the
signal processing electronics to analyze each cell. The problem isn't with
coincidence events in the sample stream that the instrument sees. Impurities
arise from cells that the instrument doesn't see (among other sources).
>I routinely do my sorts at high concentrations, cells running at 4,000 to
>5,000 per second, and consistently obtain 99.9% purity;
That depends on what percentage of cells being analyzed are being sorted.
My data shows that if the sorted population is less than 5% of those being
analyzed, running at 4-5,000/sec. can lead to 1-2% impurities. If the
sorted pop. is 1% or less and run at 4-5K/sec., the impurities can get as
high as 3-4%. Again, the sorter cannot exclude cells it doesn't see.
>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.
Getting through what? Getting through the laser while the electronics were
looking at another cell. You prove my point exactly. High flow rates result
in impurities.
>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).
Try collecting cells that would normally go to waste and reanalyze them.
You'll see what Alice Givan saw and described here on this mailing list
a year or two ago. A large number of the cells she wanted were going into
the waste task. Your yeild (number of cells in the tube you want divided
by the number of cells you get) also decreases dramatically at high rates.
>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
The word for the world is FLOW.
QUESTION AUTHORITY..............
on every fundamental precept proposed as an explanation of any
aspect of reality; because reality is a perception and therefore
inherently subjective and gaussian in distribution. (possibly Poisson)
(I said that.)
-- ============================================================================== Thomas Delohery | Internet: t-delohery@ski.mskcc.org Manager, Flow Cytometry Core Facility | Phone: (212) 639-8729 Memorial Sloan-Kettering Cancer Center | Fax: (212) 794-4019 ==============================================================================
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