Re: PE-Cy5.5 - PE compensation problem

Hi Larry,

We use these two all the time without any problem, and the only thing I  
can see that's different about your setup is the 685LP.  I'm thinking  
it may be too long- it looks like you're catching it right at the peak  
of Cy5.5 emission, so you're directing a lot of Cy5.5PE light into the  
PE detector.  Our setup is custom, and probably the reverse of yours,   
but we have a 630SP that divides the light one way for PE and PETR  
detection, and the other way for Cy5PE, Cy5.5PE, and Cy7PE.  Have you  
tried anything shorter than 685?

I just looked back, and we also used this filter when we had the  
standard Z configuration.  We've always run 200 mW 488 nm, a 580/30 for  
PE, and a 720/45 for Cy5.5 PE.

Hope that helps!

Carol


>
> 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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>
>
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Carol Oxford							
Manager, UC Davis Optical Biology Lab 	
Tupper Hall, rm 3425					
University of California				
Davis, California  95616				
(530) 752-7205							
(530) 752-4548 fax						
cloxford@ucdavis.edu
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