Ki-67 staining for determination of rhesus macaque T cell proliferative responses ex vivo
| Autor: | Matthew P. Morrow, David B. Weiner, Devon J. Shedlock, David A. Hokey, Karuppiah Muthumani, Kendra T. Talbott, Bernadette Ferraro |
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| Rok vydání: | 2010 |
| Předmět: |
Histology
medicine.diagnostic_test Cell growth T cell Carboxyfluorescein succinimidyl ester Cell Biology Biology Peripheral blood mononuclear cell Molecular biology Pathology and Forensic Medicine Flow cytometry chemistry.chemical_compound medicine.anatomical_structure chemistry Antigen medicine Fluorescein Ex vivo |
| Zdroj: | Cytometry Part A. |
| ISSN: | 1552-4930 1552-4922 |
| DOI: | 10.1002/cyto.a.20857 |
| Popis: | 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. |
| Databáze: | OpenAIRE |
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