[Cytometry] 5 color compensation issue

Mario Roederer roederer at drmr.com
Tue Feb 3 11:56:21 EST 2009


Actually, PE-Cy5.5 has roughly the same emission overlap into PE as  
does PE-Cy5 (although of course this will be lot-to-lot dependent and  
manufacturer dependent).  PE-Cy5.5 is generally better because it has  
much less compensation into the APC channel.

I'm not sure why you state that it "can't be compensated."  It  
certainly can be compensated, just as easily and "correctly" as PE- 
Cy5; just make sure to use the same reagent in your compensation tube  
as in your experiment.

As a side note, it is important to note that while fluorescence  
emission spectra give us some idea about compensation requirements,  
they are NOT very good -- this is because the FACS uses different  
amplification (gain) settings in different regions of the spectrum,  
where as a fluorescence spectrum collected from a spectrofluorometer  
will have essentially the same gain setting throughout the spectrum.

I hope that you can go back to your user and correct the  
misinformation -- and start "allowing" PE-Cy5.5 into your flow room  
again, as in many cases it is far superior to PE-Cy5.  (Incidentally,  
PerCP-Cy5.5 is even better than PE-Cy5.5 -- having less compensation  
requirement overall).

mr

On Feb 3, 2009, at 10:23 AM, Haviland, David L wrote:

> Yes.   If you compare the emission profile of PE-Cy5.5 to PE you are  
> going to find overlapping emission spectra that can't be  
> compensated.   A better choice for your user would have been PE- 
> Cy5.   I had this once myself and kindly asked the user not to bring  
> that combination in my flow room again.   It was all made clear to  
> my user when I showed her the emmission profiles of the reagents in  
> question.
>
> Hope this helps...
>
> David
>
>
>
> -----Original Message-----
> From: cytometry-bounces at lists.purdue.edu on behalf of Zhugong Liu
> Sent: Mon 2/2/2009 9:29 PM
> To: cytometry at lists.purdue.edu
> Subject: [Cytometry] 5 color compensation issue
>
> Dear all,
>
> We are running 5-color flow on FC500 and have some compensation  
> issues.
> The 5-color cocktail includes antibodies with the following
> fluorechromes: FITC, PE, PE-Cy5.5, PE-Cy7 and APC. Do you think there
> might be some reagent interactions in this panel combo, especially
> PE-Cy5.5 and other dyes? Our single controls were well compensated  
> using
> the software-generated matrices, however the fully stained samples  
> were
> not.
>
> Thanks for any comment/suggestion.
>
> Best,
> Jason
>
> -------------------------------------------------------
> Jason Liu, PhD
> Asst. Director, Flow Cytometry
> Tolerance Assays and Data Analysis
> UCSF/Immune Tolerance Network
> 3 Bethesda Metro Center, Suite 400
> Bethesda, MD 20814
> Phone: 240-235-6177
> Fax: 240-235-6198
> Email: zliu at immunetolerance.org
>
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