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> Rita describes trouble with discriminator settings on an Elite
> Unless there is a very good reason for doing otherwise, I have found it
> safer overall to discriminate only on a light scatter parameter, every
> particle scatters some light at least and junk is usually the small stuff.
I agree with Deans comments, we certainly trigger off forward scatter for cell
cycle analysis on the Elite. In our experience the width of the air cooled laser
on this machine makes doublet discrimination difficult so we are shifting most
of our cell cycle work to the XL which works better (I don't know why). On
the other hand I am surprised by Ritas result. We have triggered off
PI fluorescence in the past and had no major problem. How good is the PI
fluorescence if you trigger off forward scatter? I just wonder if the cells are all
permeabilised. When we were first setting up the TUNEL technique for
cytometry in our lab we had a lot off trouble with PI negative cells in the
tubes; fortunately this stopped when we cleaned it up using Ortho Permeafix
instead of the standard fixation/permeabilisation. Of course the rubbish was
all against the axis on a PI plot, so reasoning that an apoptotic cell (or other
leukocyte) aught to have DNA in it, we just gated out the stuff hard up
against the PI axis. Of course very late apoptotic cells can shed all their
DNA if they are not removed by macrophages first, but we figured we
could live with that. This produced the same result as triggering off PI.
As I said, the problem disappeared when we permeabilised and fixed the
cells properly, because the negative cells were not really rubbish,
they were still intact.
Have fun
...........................................................
Mike Salmon
Department of Rheumatology
The Medical School
University of Birmingham
Birmingham B15 2TT
United Kingdom
Tel: 44 (0)121 414 6780
Fax: 44 (0)121 414 6794
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