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

Autor: Shedlock DJ; Department of Pathology and Laboratory Medicine, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania 19104, USA., Talbott KT, Morrow MP, Ferraro B, Hokey DA, Muthumani K, Weiner DB
Jazyk: angličtina
Zdroj: Cytometry. Part A : the journal of the International Society for Analytical Cytology [Cytometry A] 2010 Mar; Vol. 77 (3), pp. 275-84.
DOI: 10.1002/cyto.a.20857
Abstrakt: 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.
((c) 2010 International Society for Advancement of Cytometry.)
Databáze: MEDLINE
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