I have a flow user who has limited cell availability (isolated CD34+
cells), but manages to yield about 10,000 per tube for treatment (by
microscopic hemacytometer count). She suspends the cells in a small
volume (~250 ul), and runs the tube to dry on the FC, but only is
able to collect 1500 to 3000 events ("boost" events are collected;
sample is placed on SIP very quickly, so droplet retrieval system is
not taking up a significant portion of the sample prior to
acquisition). The rate is about 10 - 15/sec on "HI" sample pressure
(FACScan). The cells are always in the same tube, and are
respuspended thoroughly. Cells are PI treated and experimentally
manipulated.
Where are 80% of the cells going every time? Is there some physical
/ fluidic / optical explanation for why they may not be counted on
the FC? Or, is there a greater chance that they may be lost prior to
acquisition; if so, how?
Please respond with even remotely possible explanations, especially
personal experience.
Thanks, Darren Hickerson
dhickerson@brody.med.ecu.edu
Core Flow Cytometry Facility
Department of Microbiology and Immunology
East Carolina University School of Medicine
Greenville, NC 27858
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