Tony, 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 ----- Begin Included Message ----- >From owner-cyto-sendout@flowcyt.cyto.purdue.edu Thu Jul 31 16:44:57 1997 X-Mailer: Novell GroupWise 4.1 Date: Wed, 30 Jul 1997 17:23:18 -0700 From: Antony Bakke <bakkea@ohsu.edu> To: cyto-inbox Subject: PI gating on FL3 -Reply Mime-Version: 1.0 Content-Type: text/plain Content-Disposition: inline Content-Length: 597 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 ----- End Included Message -----
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