On the FACScan I suggest disabling the droplet retrieval system when
running very small cell numbers.
*** Unless of course there is some biohazard potential...***
This is very easily done by removing the outer of the two concentric
tubes of the sip assembly.
Simply unscrew the nut holding the sealing ring, slide it down, and
usually the outer tube will come with it.
Store the sucker tube carefully, replace the nut, run your sample(s),
then reassemble the system afterwards.
You do need to place something like a Petrie dish to catch the drips,
else the bench gets very messy!
This may not explain your cell loss, but will eliminate one possible
cause; the sucker takes away much more than you think!
Good Luck, Joseph.
At 03:48 PM 22/10/97 -0400, 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
--
Joseph Webster
Flow Cytometry Facility
Centenary Institute
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