[Cytometry] 5 color compensation issue

Daley, John F., DFCI John_Daley at dfci.harvard.edu
Tue Feb 3 18:13:59 EST 2009

The FC500 has colinear Red and Blue laser beams with no spatial beam separation
for time delay adjustment between beams.  APC and Pe-Cy5.5 signals  will both
land on the "red"(~675nm) detector at the same time. I  believe  one has to make
use of the PE-texas red (~610nm) PMT detector  to do simultaneous  5 color
analysis  using Pe-Texas Red Tandems(aka ECD from Beckman Coulter).
John F. Daley
Director of Flow Cytometry
Hematologic Neoplasia Flow Cytometry Facility
Dana-Farber Cancer Institute
Mayer 584
44 Binney Street
Boston, MA 02115
Ph: 617-632-4571
Fax 617-582-7799


From: cytometry-bounces at lists.purdue.edu on behalf of Carol Oxford
Sent: Tue 2/3/2009 10:57
To: Zhugong Liu; cytometry at lists.purdue.edu
Subject: Re: [Cytometry] 5 color compensation issue

Hi Jason,

We run these together all the time, and all but one of our instruments
are set up to run this combination- we even add PETR and PE-Cy5 to the
mix.  I'm not familiar with the exact filters on the FC500, it's
possible that changing the filter setup might improve things.  If you
run the appropriate compensation controls and the FMO controls, you
should be able to get this to work.  It takes a bit of panel
optimization to make sure that your positive population is well
separated and above the spread of the other dyes in the mix, but it
routinely works well for us.


Zhugong Liu wrote:
> Dear all,
> We are running 5-color flow oFC500 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
> _______________________________________________
> Cytometry mailing list
> Cytometry at lists.purdue.edu
> https://lists.purdue.edu/mailman/listinfo/cytometry


Carol Oxford                                          
Manager, UC Davis Optical Biology Lab      
Tupper Hall, rm 3425                            
University of California                         
Davis, California  95616                       
(530) 752-7205                                                    

Manager, Stem Cell Sorting Facility
(916) 703-9307

cloxford at ucdavis.edu
http://ccresources.ucdmc.ucdavis.edu <http://ccresources.ucdmc.ucdavis.edu/>


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