>
>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
Aliens. They took them. Sucked them right our of your machine with a
great big straw.
To remote? Sorry.
Are you sure that your droplet containment module isn't sucking them
away? 250ul isn't that much fluid. You might suggesting that she
disenable the DCM by just removing the outer sheath.
Another possibility...Is it possible that most of the cells are in that
initial burst you get when you put a tube on the machine? If so, they
might be lost electronically from the time it takes from putting the
tube on until you hit acquire. A suggestion there would be to have the
instrument already acquiring before you put the tube on. Just some shots
in the dark.
I personally like the alien theory. What do you think: The FACS
Conspiracy...well it might bot be a blockbuster, but it could be a great
X-Files episode.
I'll stop now.
Bill.
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