[Cytometry] 5 color compensation issue

Haviland, David L David.L.Haviland at uth.tmc.edu
Tue Feb 3 15:28:50 EST 2009

Well, I'm not here to start an arugment but I only know from my own
experience which mirrors what I see when comparing PE, PE-Cy5.5, and
PE-Cy5 on the BD spectral viewer.   In closing everything down on the
spectral viewer except the emission profiles of those three, it would
appear that PE-Cy5.5 has a very strong emission, virtually covering the
PE emission making the two very difficult to compensate.  Per the
spectral viewer, PE-Cy5.5 has two emission peaks, one about 550 and the
other short of 700nm.  PE-Cy5, however, only has maybe a 5% emission in
PE making those two workable. 

I don't know if it will come over by I've "snipped" and included the
figure from the BD viewer showing the emission profiles of the three.
I don't know about others but I have the standard filter configurations
in my LSR and Aria (both 10 color) and my user called me over when they
couldn't comp PE and PE-Cy5.5 against each other.   When I had
difficulty, we both went to the viewer to double check after taking the
respective comps to 900+ without resolution.   Said user has since
changed the flours they use.   

Understand that my not "allowing" the pair in the room was pretty much
in jest as well. Seeing the overlap in PE and PE-Cy5.5 in the PE channel
on the spectral viewer pretty much convinced my user to find another
color on their own.   Many users get Cy5 and Cy5.5 confused. 

Won't argue about PE-Cy5/Cy5.5 and APC.   I've dealt with them as well
but at least the combinations were "workable". 


David L. Haviland, Ph.D.
Assistant Professor, Immunology
Institute of Molecular Medicine, R637I
Univ. of Texas, Houston - HSC
1825 Pressler
Houston, TX  77030
713-500-2413 - Voice
713-500-2424 - FAX
-----Original Message-----
From: Mario Roederer [mailto:roederer at drmr.com] 
Sent: Tuesday, February 03, 2009 10:56 AM
To: cytometry at lists.purdue.edu
Cc: Zhugong Liu; Haviland, David L
Subject: Re: [Cytometry] 5 color compensation issue

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).


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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