APC-Cy7 and PE-Cy7 compensation

Oscar Fornas oscar.fornas at upf.edu
Tue Nov 11 05:05:28 EST 2008

Hi Lynda,

The phenomenon that you describe is completely normal using single color
compensation samples.

For the polychromatic flow cytometry 2 and 3 color compensations controls
should be performed for proper color compensation.
The automatic color comp in DiVa software can be good enough for 2 colors
analysis, but using more than 4 colors this tool not always is enough for a
proper compensation and you must set it manually with additional controls.

When using several antibodies in a sample, the spreading of negative cells
for a concrete antigen can be rise since autofluorescence increases giving
high spillover in other detectors due the addition of a lot of antibodies in
the same sample.
This means that compensation with 2 and 3 color comp controls is mandatory,
otherwise this affect to the spillover and can concludes with a under or
over compensation. Furthermore, manual color comp must be performed !!!

This phenomenon that all of us identifies and correct with additional color
compensation with 2 and 3 color controls was finally fully described and
illustrated by Mario Roederer (in my opinion) in one of the best technical
flow cytometry papers. Check it for an amazing guide of color compensation.
Reference is:
Optimizing a multicolor immunophenotyping assay
Clin Lab Med. 2007 Sep;27(3):469-85, v. Review

Hope this helps.

Good luck!!!



Òscar Fornas, PhD. 
Technical Director, Flow Cytometry Facility. 
DCEXS Universitat Pompeu Fabra. 
Barcelona Biomedical Research Park. 
Doctor Aiguader, 88. 
08003 Barcelona. 
Phone:+ 34 93 316 08 50 
Fax: + 34 93 316 09 01 
E-mail: oscar.fornas at upf.edu 
Web: http://www.upf.es/cexs/sct

-----Mensaje original-----
De: Guzik, Lynda [mailto:GuziLJ at ccm.upmc.edu] 
Enviado el: viernes, 07 de noviembre de 2008 22:39
Para: Cytometry Mailing List
Asunto: APC-Cy7 and PE-Cy7 compensation

Hello All,

I have a researcher who would like to do 6color flow using FITC, PE, PI,
APC, APC-Cy7, and PE-Cy7.  We are using a FACSVantage DiVa with a 488nm,
635nm and UV lasers.  We are having problems with compensation between the
APC-Cy7 and PE-Cy7 channels.  Using single color positive controls and the
DiVa algorithm, the compensation looks fine.  However, when we put APC-Cy7
and PE-Cy7 antibodies in the same tube, the plots look like there is extreme
overcompensation of PE-C7 against the APC-Cy7 channel.	I've attached a
couple of plots to illustrate.	I am able to correct for this, but wonder if
I should be.  And I fail to understand why the single color controls look
fine but have a problem with the dual stained tube.  Any input would be

Thank you,
Lynda Guzik
McGowan Institute - Lagasse Labs
Rm 520A - Bridgeside Point Bldg
100 Technology Dr - Suite 200
Pittsburgh, PA 15219
guzilj at upmc.edu<mailto:lguzilj at upmc.edu> or
lyg1 at pitt.edu<mailto:lyg1 at pitt.edu>

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