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[BSM]

I am hoping to reach someone who is familiar with this assay. In brief, you pulse splenocytes with a peptide that is expressed, in my case, from an active viral infection. These splenocytes are then labeled with CFSE at a higher concentration. A separate pool of splenocytes are pulsed with no peptide or an unspecific peptide with a lower CFSE concentration. The two populations are mixed and then adoptively transferred into a mouse who was previously infected with a virus. The hope is that this mouse CD8 CTLs will kill donor APCs that were pulsed with the peptide. This will result in a loss of your CFSE high population when analyzed via FLOW. The problem I have recently encountered is that my CFSE high donor splenocytes do not appear as a discrete population in CFSE high. Rather, the histogram peak is more spread out compared to my CFSElow control population on the histogram plot. Any ideas as to why this is occuring?

For example if you look at this figure (non immunized primary plot)both populations look the same.

http://www.nature.co...ml#figure-title


However, mine looks like this:




Both of my population % look good. It just doesn't look good visually