GFP expression queries

From: lklee@rci.rutgers.edu
Date: Fri Oct 31 2003 - 16:11:48 EST 

Greetings, I use the FACSCalibur and have a couple of questions:

 

1.	I normally use PBS (w/o Mg2+ and Ca2+) with propidium iodide as
resuspension buffer for analysing GFP expression in mammalian cells. Is it
necessary to fix the cells in paraformaldehyde if I analyse them immediately
after detachment from their wells? I presume that since I am also excluding
the dead population using propidium iodide, fixing would kill the cells and
hence affect the quantification of live GFP-positive cells.

 

2.	I do not see a distinct narrow peak for my GFP plasmid transfected
cells. It is more like a small peak with a wide distribution (green overlay
in attached picture). I have adjusted the FL-1 voltage according to the FL-1
fluorescence of untransfected cells (filled purple histogram). Is it normal
for the GFP expression to span such a wide intensity range?

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