[Cytometry] 5 color compensation issue

Victoria Best bestv at ccri.net
Mon Feb 2 23:50:58 EST 2009


Hi Jason,

Actually, had recent experience with an eerily similar scenario.

Was using FITC PECy7, PECy5.5, APC and Alexa700 together.  For me Single
color compensation controls only told half the story, but samples stained
with every color except one (fluorescent minus one control) could sort out
who was the troublemaker.  The trouble was with the 600-700 emitters.
Caused by APC bleed over.

Additionally, that some of my markers are "dim", "intermediate" and
"bright", and all expressed together on the same population of cells
certainly didn't help.

In the end I threw out Alexa700 and flip-flopped the markers on PECy7 and
PEcy5.5.  

I'm sorry if you've already ruled out compensation issue- that really was my
issue. I now have a working panel that uses all 5 of these colors.

Hope that was helpful,
Victoria. 



  




On 2/2/09 10:29 PM, "Zhugong Liu" <zliu at immunetolerance.org> wrote:

> 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
> 
> _______________________________________________
> Cytometry mailing list
> Cytometry at lists.purdue.edu
> https://lists.purdue.edu/mailman/listinfo/cytometry

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