It is important here to note Dave Coder's point. The PE signal that gets
into the PI channel *is* significant and important to compensate. Otherwise, a
user may gate out very bright PE as low PI, and throw out bright PE
expressors as 'dead' by mistake. New users have done this many times, with both
low (< 1.0ug/ml) and high (5ug/ml) concentrations of PI. Using low amounts of
PI makes the FL3 (PI) minus FL2 (PE) compensation even more critial to proper
live cell/dead cell discrimination, since the 'dead' signal is often closer to
background than with higher concentrations of PI, and chances of getting bright
PE into low PI ranges goes up.
Regards,
Joe Trotter
Director, Flow Cytometry
The Scripps Research Institute
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Date: Wed, 30 Jul 1997 17:23:18 -0700
From: Antony Bakke <bakkea@ohsu.edu>
To: cyto-inbox
Subject: PI gating on FL3 -Reply
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PI can be used to gate out dead cells in combination with PE and FITC staining
(see Sasaki et al, Cytometry 8:413-420, 1987). It is probably advisable to use
a low concentration of PI. Sasaki used 0.5 ug/ml, but I have seen protocols
using up to 5 ug/ml, but not the higher concentration of 50 ug/ml used for cell
cycle staining. Since you are gating out the PI positive cells and analyzing
the negative cells, the spectral overlap between PI and PE is not significant.
Hope this helps,
Tony Bakke, Ph.D.
Director, Clinical Immunology and Flow Cytometry Lab
Oregon Health Sciences University
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