RE: Comp beads and negative populations

To all,

We only run the unstained COMP bead as our negative controls, autofluorescense has little
or NO effect on compensation. You can empirically test this by setting up COMPS both
ways. Cell autofluorescense is different for different lasers the beads do not, except in
the near UV range. 

To properly set compensation, you must ensure that:
(1) Your compensation control is at least as bright as your stained sample
(2) When, for any given fluorochrome compensation control, you are 
comparing a positive to a negative (or dim) population, both 
populations have the same autofluorescence (e.g., both + and - are 
macrophages, or both are lymphocytes, or both are beads)
(3) You do not change any instrument settings such as PMT voltages of 
the fluorescence channels between collecting the compensation control 
and collecting your sample.

The standard COMP beads (kappa-antibody labelled) do have limitations where cells will be
required, such as the use of interculating viability dyes or CFSE. In addition, they can
not capture lambda antibodies.

SP



=======================================================

Stephen P. Perfetto, MS.,MT. (ASCP)
Manager, Core Flow Cytometry Facility
Vaccine Research Center, NIH
Building 40
40 Convent Dr., Room 5507
Bethesda, MD 20892-3015

email: sperfetto@nih.gov
Phone: (301) 594-8659

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> ----------
> From: 	Rick Dunham
> Sent: 	Tuesday, November 29, 2005 6:10 PM
> To:	Cytometry Mailing List
> Subject:	Comp beads and negative populations
> 
> Hello list,
> 
> We recently started using Ig capture beads for compensation and are  
> in a debate over whether to use unstained cells or unstained beads as  
> the negative population in automatic compensation with DiVa or	
> FlowJo.  One 'school of thought' is that the positive and negative  
> populations should have the same autofluorescent properties, thus  
> dictating that one should use unstained beads as the universal	
> negative population.	The other 'school of thought' is that the  
> negative population should have the same autofluorescent properties  
> as the sample, dictating that one should use unstained cells as the  
> universal negative population.	Both arguments seem to have merit,  
> though looking at previous discussions of autofluorescence and	
> compensation on this list and in BD and FlowJo literature,  
> autofluorescense is thought to have little effect on compensation.
> 
> However, one can imagine a scenario from the 'old days' of manual  
> compensation when for many, the goal was equalization of the median  
> of the positive and negative populations for a given stained channel	
> vs the other unstained channels, in which different autofluorescent  
> properties of the experimental sample vs the compensation sample  
> would generate over or under compensation, if the mean	
> autofluorescence of one channel of the negative events were  
> significantly different with respect to another channel.
> 
> This is where we are in this discussion.  Does anyone out there in  
> the ether have a solution or any comments?  What do you do when using  
> beads?
> 
> Rick Dunham
> Graduate Student
> Emory Vaccine Center
> Emory University
> Atlanta, GA USA
> 
> rdunham@emory.edu
> 
>