Darren,
That is an interesting problem. My guess is that the cells are lost
primarily in the droplet retrieval system. There are 2 easy ways to
check this.
1. Run the cells in a larger volume (ie: 1 ml). It will take a long time
to run, but, if the cells are being lost in the droplet retrieval system
during the first few seconds, then a larger sample volume should reduce
the loss of cells.
2. Remove the droplet retrieval tube by turning the retaining ring and
slide the outter tube down and off. The sample tube will drip between
samples, but this will eliminate the droplet retrieval system.
If the above make no difference in the number of cells counted, then the
cells are most likely lost before the tube is put on the cytometer.
If #1 fixes the problem, but #2 does not, then my guess is wrong and the
problem is deadspace volume (ie: not all of the sample reaches the flow
chamber).
Good luck,
Roy
--
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| W. Roy Overton, Ph.D. |
| overton@umdnj.edu |
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Darren Hickerson wrote:
>
> 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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