Re: GFP sorting question

From: Larry Arnold (lwarma@med.unc.edu)
Date: Fri Oct 18 2002 - 08:48:04 EST 




Andy


What type of cells are the GFP ones that are giving you the
problem?  Are they PBLs like your test sample?  I'm betting
that they are a different cell type that has a size or shape that is not
compatible with the diameter sorting tip (70um?) you are using.  It
is very unlikely that it is the sample preparation i.e the GFP itself,
the media etc.).  I see this problem regularly and it is always
solved by using a larger diameter tip.


Larry


At 04:45 PM 10/16/2002 -0400, you wrote:


Hi FLow-er in Flow land,

I've come across an interesting (and confusing) problem which sorting GFP
cells.  I've noticed several times that I get alot of fanning while
sorting.  I don't believe it is the Frequency, drive or phase since
setting up the sort gives me beautiful side streams and great
separation.  I first notice this on my Elite and now I have a
FACSVantage and 2 of the last 3 GFP sorts showed this problem. 
After todays sort, which had terrible fanning, I put on a sample of
unstained fixed PBL's to see if it was instrument related.  Not
surprisingly, that sample gave great side streams.  Obviously the
problem is with the sample.  I've never (never say never!) seen a
fanning problem with surface stained cells and I don't understand what
could be causing this to happen with GFP cells?  Could the
transfection method somehow "charge" the cells which interferes
with the charge applied for sorting?  As far as I can tell the media
(which is MEM with Pen, strep, L-Glut, FCS, bicarb.) shouldn't do
anything?


Any help or suggestions would be greatly appreciated!


Thanks in advance.


Andy


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Lineberger Comprehensive Cancer Center

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