Darren:
I suggest the user stains/puts her samples in T1000 tubes supplied by
MARSH (800-445-2812). These tubes will hold about 2ml. They also slide
inside the 12x75 tubes used on the scan. You can take the sample
containment tube off the scan as well. This will leave just the sample
injection rod. The containment system will waste some sample. WE take
it off for samples less than 200ul.
Some of the cells are lost due to washing steps. Mininmize wash to only
2. You can try 1 but the background may be higher. Try
staining/washing in the above tubes.
Jim Houston
> -----Original Message-----
> From: Darren Hickerson [SMTP:dhickerson@brody.med.ecu.edu]
> Sent: Wednesday, October 22, 1997 8:48 PM
> To: Cytometry Mailing List
> Subject: Lost cells
>
>
> 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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