sorting EGFP transfectants

Dear flowers:
First and foremost: Happy new year!
I have a problem interpreting some data.
I transfect 293 with an eukaryotic expression plasmid
coding for EGFP. EGFP sequence is located downstream
of an IRES site. After selection with G418 I get about
10% stably transfected cells. I tried sorting the high
1% but these cells did not grow. Since the
fluorescence from EGFP is largely overlapping with
autofluorescence I tried to sort the cells fluorescing
in green but not in red using non-transfected 293 as
compensation control. The enclosed JPG shows the
setup, R2 is the sort gate. Since I cannot do a post
sort control (too few cells, too slow sorter, too
little time....) I check the cells after they have
grown up again. Compared to the wildtype 293 I see a
shift in the histogram, but it is the whole "diagonal"
that has shifted when I look at the dot plot. Now here
is the question: Can I consider these cells positive
for EGFP? Or is something else happening that leads to
a "shift of autofluorescence"? I had expected to see
something more cloudlike, similiar to the appearance
when sorting.

Thanks in advance

Jan

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Jan Mueller-Berghaus, MD
Skin Cancer Unit of the German Cancer Research Center
http://www.dkfz.de/melanom
http://www.aargh.onlinehome.de/burgh/burgh00.htm

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