[Cytometry] 5 color compensation issue

Mario Roederer roederer at drmr.com
Wed Feb 4 06:17:02 EST 2009


While it is well-known that tandems have some stability issues when  
exposed to light or fixative, it is important to point out that this  
can only "screw up compensations" if you are not creating compensation  
controls properly!  (Also, as others have noted, it is important to  
recognize that tandems do not, on their own, "become unstable."  It is  
a very predictable property based on light exposure for PE tandems, or  
extended exposure to fixative for some APC tandems.)

As long as you treat your comp control identically to your sample, and  
you are using the same reagent to compensate as you are in your  
experiment, it really doesn't matter if the tandem has started to  
deteriorate -- because the compensation control will have the same  
spillover as the experimental sample.

Sometimes users try to use one Cy7PE reagent to compensate for  
multiple different Cy7PE reagents -- this will often NOT work.  Every  
tandem should generally be treated as a different color (and require  
its own compensation control).  Of course, if you prove (by, for  
example, examining the resulting compensation matrices from each  
tandem) that the spillover values are the same for different tandems,  
then you CAN use one to compensate for multiple.

Some software packages will not allow you to have multiple  
compensation settings in a single experiment.  Others will.  If you  
use different tandems in the same experiment, then you will need to  
consider using a software package that can allow for multiple  
compensation matrices in the same experiment.

mr

On Feb 3, 2009, at 9:19 PM, Michelle Tourigny wrote:

> We have had many problems with PE-Cy7 becoming unstable and screwing  
> up
> the compensations.
> michelle
>
> Michelle R. Tourigny, Ph.D.
> Research Assistant, Instrument Specialist
> Telethon Institute for Child Health Research
> 100 Roberts Road, Subiaco
> Western Australia, 6008 AUSTRALIA
>
> Ph 61 8 9489 7878, Fax 61 8 9489 7700
> email michellet at ichr.uwa.edu.au
>
>
> -----Original Message-----
> From: cytometry-bounces at lists.purdue.edu
> [mailto:cytometry-bounces at lists.purdue.edu] On Behalf Of Carol Oxford
> Sent: Wednesday, 4 February 2009 12:57 AM
> 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.
>
> Carol
>
> 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
> CBST
> (916) 703-9307
>
> cloxford at ucdavis.edu
> http://ccresources.ucdmc.ucdavis.edu
>
> ------------------------------------------------------
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