Peripheral blood mononuclear cells (PBMC) were prepared by density-gradient centrifugation (Lymphocyte Separation Media; ICN Biomedicals). Viably frozen cell suspensions were analyzed on a LSRFortessa or a FACSCanto II flow cytometer (BD Biosciences). Cell populations were gated on forward scatter and side scatter. For T cell analyses, additional CD3, CD4 and CD8 gating was used with a minimum of 50,000 CD4+ T events being collected. For analysis of proliferation, intracellular staining for Ki67 was performed. Additionally, surface staining for the activation markers HLA-DR and CD39 as well as PD-1 was performed (all antibodies from BD biosciences). Cell populations were identified as previously described and expressed as percentages of the parent population.(33 (link)) The human Th17 subset of CD4+ T cells was identified as CXCR3 negative and CCR6 positive.(34 )
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Characterization of Human Th17 Cells
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Antibodies
Ccr6
Cell
Cxcr3
Density gradient centrifugation
Frozen
Hla dr
Human
Intracellular
Lymphocyte
Parent
Peripheral blood mononuclear cells
T cell
T cells subset
Th17
Corresponding Organization : National Heart Lung and Blood Institute
Other organizations : Copenhagen University Hospital, Rigshospitalet, National Institutes of Health Clinical Center, National Human Genome Research Institute, Pharmacyclics (United States), Center for Cancer Research
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Variable analysis
independent variables
- Density-gradient centrifugation to prepare peripheral blood mononuclear cells (PBMC)
- Cell populations gated on forward scatter and side scatter
- T cell analyses, including CD3, CD4 and CD8 gating
- Proliferation of T cells, measured by intracellular staining for Ki67
- Expression of activation markers HLA-DR and CD39, as well as PD-1
- A minimum of 50,000 CD4+ T events being collected for analysis
- No positive or negative controls were explicitly mentioned in the provided information.
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