Ki-67 staining for determination of rhesus macaque T cell proliferative responses ex vivo.
Cytometry A
; 77(3): 275-84, 2010 Mar.
Article
em En
| MEDLINE
| ID: mdl-20104580
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.
Texto completo:
1
Coleções:
01-internacional
Base de dados:
MEDLINE
Assunto principal:
Linfócitos T
/
Antígeno Ki-67
/
Citometria de Fluxo
Limite:
Animals
Idioma:
En
Revista:
Cytometry A
Ano de publicação:
2010
Tipo de documento:
Article
País de afiliação:
Estados Unidos