Wayne,
The general rule of thumb is to have the particle diameter not exceed
roughly 1/4 the stream diameter. If it does, the recovery drops rather
dramatically because droplet breakoff is affected by the large particles.
You may do much better with MacoSort and a larger nozzle. For
high recovery we usually go no more than 1/5 the nozzle diameter as
the upper limit for particle size. We frequently use the 70, 80, 90,
and 100 um nozzles on our systems depending on cell size. The
200um (MacroSort needed) may be the best one here for recovery, but
your sort rate will have to drop in phase with your drop generation
rate - which will be relatively slow with a 200um nozzle. So, you're
going have to go slow to get high recovery with particles that large.
Joe
-----Original Message-----
From: Wayne Green <Wayne.Green@hci.utah.edu>
To: cyto-inbox
Date: Tuesday, March 09, 1999 11:50 AM
Subject: Sorting C. elegans embryos
>
> An investigator here wants to sort C. elegans embryos expressing
>GFP. The embryos are anywhere between the 28 cell to 600 cell stage with
>most closer to the smaller size. Each embryo is roughly 50x30 microns,
>ellipsoid in shape with a fairly rigid shell encasing the embryo. We are
>told that the embryos are relatively resistant to physical trauma.
> We have run these on a Vantage with a 100 micron tip (no Macro Sort)
>and can detect the GFP expressing embryos. Our problem arises with the
>sorting; if we sort 40,000 GFP positive embryos, one only finds about 100
in
>the collection tube. The good news is that those 100 are healthy in that
>they grow normally following the sorting process. The question is,
>what might cause this abysmal sorting efficiency? When we sort "normal"
>cells like murine stem cells, lymphocytes, etc. we get very good recovery
>and purity. If there is anyone out there with experience sorting something
>like this I would greatly appreciate your suggestions.
>
>Thanks
>
>Wayne F. Green, Ph.D.
>HCI Flow Cytometry Core Lab
>wayne.green@hci.utah.edu
>
>
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