Re: PE-Cy5.5 - PE compensation problem

Larry,

The difference between the MoFlo and the Calibur may be due to the 
relative PMT settings you have on each instrument.  This is a function of 
the filter sets used on each instrument.  The problem is probably with the 
"PE-Cy5.5 filters" not the PE filter.  You are using a broad 650LP FL3 
filter on the Calibur.	From your note the filter for the PE-Cy5.5 is much 
narrower on the MoFlo; 700/50 ( probably using it with a PE-Cy7 channel 
too).	I also suspect that since the Calibur is a cuvette system you are 
getting much more of a signal compared to a stream in air system like a 
MoFlo (or a Vantage).  One way to tell if this is true is to look at your 
PE into PE-Cy5.5 channel spillover.  If it is significantly higher on the 
Calibur than the MoFlo that the likely problem.  Try using the exact same 
filters for each instrument and see if the problem exists.

Alternatively (or in addition) it might be due to the fact that you are 
using a high powered laser on the MoFlo and a low one on the Calibur.  PE 
tends to level off or get duller at high laser powers (>100mW); however 
the tandems love the higher laser power and get brighter with more power. 
Thus on the MoFlo you may have increased the PMT of the PE channel to 
compensate for the PE saturation.  This would lead to the situation you 
describe.

these differences in spillover are a problems when comparing data between 
any two different instruments.

Good luck,

Alan

Alan Stall
Director, R&D
BD Biosciences
--------------------------------




Larry Arnold <lwarma@med.unc.edu>
05/26/05 04:02 PM
	To:	Cytometry Mailing List <cytometry@flowcyt.cyto.purdue.edu>
	cc:	(bcc: Alan Stall/SDCA/BDX)
	Subject:	PE-Cy5.5 - PE compensation problem


I am having a problem on my MoFlo that I can not figure out. When I run 
PE-Cy5.5 I am getting a huge amount of signal in the PE channel (or maybe 
too little in the PE-CY5.5 channel). So much that I can not compensate 
properly - needs about 60-70% and then still doesn't look right. When I 
run 
the same sample on the FACSCalibur it looks great and only needs 12% 
compensation. Thus the problem is not the reagent. I have considered the 
filters as a problem but 2 different PE filters gave the same result. I 
have used a 580/30 and a 575/25. These filters in front of the PE detector 

in all other fluorochrome combos that I have tried give no problem.  Other 

parameters are 200mW 488nm, 685LP dichroic and a 700/50 filter in front of 

the PE-Cy5.5 detector.		 I have not tried other filter combos in 
front of 
the PE-Cy5.5 detector.		 Anybody have any ideas? Thanks

Larry


Larry W. Arnold, Ph.D.
Research Professor  and Director, Flow Cytometry Facility
Department of Microbiology and Immunology
Lineberger Comprehensive Cancer Center
CB# 7290
University of North Carolina at Chapel Hill
Chapel Hill, NC 27599
Phone: 919-966-1530
FAX: 919-962-8103 





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