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