I'm having a problem with PE and APC antibodies. I am doing four color analysis of peripheral blood lymphocytes, two tubes are stained with CD45RA Fitc, CD62L PE. CD3 PerCP and CD4 or CD8 APC. I gate on CD3+CD4+ or CD3+CD8+ then look at the CD45RA CD62L plot to measure the number of naive cells. The problem is that the CD62L staining is much dimmer when gated on CD3+CD8+ than with CD3+CD4- although this is essentially the same population of cells. This effect is most pronounced with dim CD8 APC antibodies, I get the same effect but less so with CD4 APC. Even harder to explain is the fact that I don't see this effect in all individuals. Could the APC be absorbing some of the PE fluorescence? Steric hindrance? Whats going on? Has anyone else seen this? I'm using a FACS Calibur with 4th color, I would just gate on the CD4 negatives but this would mean I would include gamma/delta cells which are quite numerous in some individuals. Any suggestions welcome Simon Monard Aaron Diamond Center New York
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