[Cytometry] 5 color compensation issue

Victoria Best bestv at ccri.net
Tue Feb 3 11:18:25 EST 2009

I use PE with PEcy5.5.  And it works.  So perhaps the choice of filter sets
matter?   Our long-pass filters are quite strict running off the 488 laser.

My advice would be to use FMOCs, on whatever configuration your machine
happens to have, and see which of your color/colors do not work together.

If taking into account all factors- machine configuration, filters, markers
you are putting your colors on, etc., I'm getting the idea that broad
sweeping advice about these 5 colors in question may not get you very far.

Sorry, needed to chime in again once more.

Good luck,

On 2/3/09 10:23 AM, "Haviland, David L" <David.L.Haviland at uth.tmc.edu>

> 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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