LARGE CELL SORT

Geoffrey Osborne geoff at sparc.brc.ubc.ca
Tue Apr 4 13:44:18 EST 2000


Hi Randall,
	My experience with large nozzle sorting has been with the mostly
the 300um and occasionally the 400um nozzle. My advice is to obtain as
"clean" a preparation of the particles of interest as possible. While this
may seem obvious, the importance of maintaining cells in a single cell
suspension and not having them stick together is critical in obtaining a
stable stream breakoff. This is often difficult due to the effect of
gravity on large particles in suspension, as clumps of 2 or more cells
really become a problem.
	 On the Vantage SE I'm unable to sort  really large stuff (~200um
organisms from termite guts), as it seems to be beyond the normal rules of
thumb that one would normally use with respect to particle size and nozzle
selection,  but particles up to about 100um are  "do able". For your
particle size you may actually get better stability from the system by
using the 400 over the 300um nozzle. For the 400um nozzle I'd be looking at
about 1000hz ddf, 2PSI and slow flow rate. Block off any other waste
aspiration that you've got on the system as you want to draw as much waste
from the main aspirator as possible. Remember also that you may need to use
the different obscurator bars, and change ND filters on the FSC diode too.
	My last tip is be patient, and persist. Hope some of this helps
Geoff


>Dear All,
>
>We have some large cells (70-80um) to sort on our new FACSVantage, and
>have no experience
>with cells of this size. Before we run through gallons of sheath, we would
>like to
>hear of your experience with large cell/large tip sorting.  It looks like
>the 300um
>tip is recommended for this size cell.
>
>The Vantage is running well on normal 70um tip for blood and cultured
>cells.  (Given the
>problems that occur with a new machine).
>
>All advice Welcome!
>
>Thanks
>
>Randall Smith
>Oregon Health Sciences University
>Flow Cytometry Core Facility
>Portland, Oregon


=====================================================
Geoffrey Osborne
Manager, Multi User Flow Cytometry Facility
Biomedical Research Centre,
2222 Health Sciences Mall,
University of British Columbia
Vancouver B.C V6T 1Z3 CANADA
Email: geoff at brc.ubc.ca
Phone: 604 822 7838
Surfing the Web?: Try http://jcsmr.anu.edu.au/facshome.html or
http://www.cytometry.brc.ubc.ca
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