biostudies-literatureShedlock DJNIAID NIH HHS275-84https://www.ebi.ac.uk/biostudies/studies/S-EPMC2939446biostudies-literatureUnknown77(3)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.Cytometry. Part A : the journal of the International Society for Analytical CytologyKi-67 staining for determination of rhesus macaque T cell proliferative responses ex vivo.PMC2939446T32 AI070099R01 AI092843U19 AI048241-07U19 AI048241Morrow MPHokey DAWeiner DBShedlock DJTalbott KTFerraro BMuthumani KfalseKi-67 staining for determination of rhesus macaque T cell proliferative responses ex vivo.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.2010-01-01T00:00:00Z2010 Mar2020-10-31T08:22:55Z2019-03-27T00:34:06ZS-EPMC29394462010458010.1002/cyto.a.20857