Ki‐67 staining for determination of rhesus macaque T cell proliferative responses ex vivo

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Abstract
The capacity for robust proliferation upon re‐infection is a hallmark of adaptive immunity and the basis of vaccination. A widely used animal model for the study of human disease is the rhesus macaque (RM), where capacity for proliferation can be assessed ex vivo using carboxyfluorescein succinimidyl ester (CFSE)‐based dilution assays. However, we show over the course of the standard ex vivo proliferation assay that CFSE‐labeling at commonly used dye concentrations induces significant cell death, but that this phenomenon is dose‐dependent. Here, we describe an alternative semiquantitative method for estimating T cell proliferative responses that avoids the putative biases associated with chemical modification. RM peripheral blood mononuclear cells were stimulated ex vivo with cognate peptides for 5 days, immunostained for intracellular Ki‐67, and then analyzed by flow cytometry. We describe a gating strategy using Ki‐67 and side light scatter, also a marker of blastogenesis, which correlates strongly with data from CFSE dilution. We show that this method is a valid tool for measuring RM antigen‐specific cellular proliferation ex vivo and can be used as an alternative to CFSE dilution assays. © 2010 International Society for Advancement of Cytometry

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