Re: Sorting big cells on FACSAria!

The cells are disturbing the stream.  There is a limit to the maximum size
of the cells with the nozzle size.  I can't seem to remember, but I think
it is somewhere around 70 % of the nozzle size.  We do large cells using a
100 uM nozzle and get pretty good results.  We also have a 130 nuzzle which
also provides good results with very large cells but slows the throughput
considerably.

Accudrop beads work just as well with the 100 uM nozzle.  We have had no
contamination issues with the sample following Accudrop use.  We simply run
a bit of bleach through the sample tubing.  The beads do not contaminate
your sheath flow.  You may also wish to run the sample line back flush for
a few minutes to be sure you have removed most of the beads.  Most sorts we
have done appear to be "clean" following sorting.  Ask your investigators
if they routinely use antibiotics in their culture medium following sorting
and replating.	This usually takes care of any "stray" bugs you may pass
through the sample tubing.

Charles A. Kuszynski, Ph.D.
Associate Professor
Director, Cell Analysis Facility
University of Nebraska Medical Center
985816 Nebraska Medical Center
Omaha, NE 68198-5816
402 559-6299 office
402 559-6267 lab
402 980-7654 cell
402 559-4069 fax
ckuszyns@unmc.edu

"What a long strange trip it's been" Grateful Dead

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	     vhodara@sfbr.org						   
	     05/07/2008 03:58						To 
	     PM 		       Cytometry Mailing List		   
				       <cytometry@flowcyt.cyto.purdue.edu> 
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								   Subject 
				       Sorting big cells on FACSAria!	   




Dear experts in flow sorting:

I am trying to sort HUH7 cells GFP (brigth and not so bright populations)
Just
two gates in a single color FL1 vs SSC (very simple sorting should be).
Those
are big cells so I tried with the 70um nozzle but with medium pressure. I
haven't had problems with settings, or with accudrop. However, I couldn't
have
pure populations. When setting the streams I can see the two perfect dots
on
the camera on the screen and accudrop run perfectly, but when I am sorting
I
don't see but a smear of white dots to the right and to the left of the
main
stream (is that because of the big cells?). I am planning to try the 100um
nozzle, but can I run the accudrop with this nozzle eventhough they are
very
small? If somebody can give me some tips, I will be very grateful.
And another question, if I prepare the machine for asceptic sorting, can I
run
the accudrop before or I need to prepare a sterile solution of these beads
or
run them after asceptic cleaning and then run again bleach and water for a
few
minutes to keep the "sterility". (We do not have the FACSAria in a BSL-3
lab
yet)
Thanks a Lot
Vida