Flow cytometry of CD4+ T cells was performed as previously described (7 (link)). The following antibodies were used for T cell phenotyping: CD45 APC-Cy7 (Biolegend), CD4 APC (Biolegend), FoxP3 PE (Thermo Fisher/eBioscience), RORγt PerCP-Cy5.5 (BD Bioscience), and IL-17A-PE (Biolegend), and dead cells were excluded using Zombie NIR (Biolegend). To detect intracellular IL-17A, isolated lymphocytes were first restimulated with 5 ng/mL phorbal 12-myristate 13-acetate and 500 ng/mL ionomycin in the presence of monensin (Biolegend) for 3.5 h. FoxP3 and RORγt were analyzed in unstimulated cells. The Vβ chain repertoire analysis used the following antibodies: Vβ2 AF647 (Biolegend), Vβ3 BrilliantViolet 510 (BD), Vβ5.1/5.2 PE-Cy7 (Biolegend), Vβ6 BrilliantViolet 650 (BD), Vβ7 FITC (Biolegend), Vβ8.1/8.2 APC-Vio770 (Miltenyi), Vβ10b BrilliantViolet 711 (BD), Vβ11 BrilliantViolet 421 (BD), Vβ12 BrilliantViolet 480 (BD), Vβ13 PerCP-eFluor710 (ThermoFisher/eBioscience), Vβ14 biotin (ThermoFisher/eBioscience) with streptavidin Qdot800 (ThermoFisher), Vβ17a BrilliantViolet 605 (BD), CD45 BrilliantViolet 750 (BD), FoxP3 PE (ThermoFisher/eBioscience) RORγt APC (BD), and CD4 BrilliantViolet 570 (Biolegend). T cell phenotyping data and Vβ chain phenotyping data were acquired using an Aurora spectral cytometer (Cytek) and data were analyzed using SpectroFlo and FlowJo 10 software.
Protocol detail
Flow Cytometry Analysis of CD4+ T Cells
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CD45 APC-Cy7
CD4 APC
RORγt PerCP-Cy5.5
IL-17A-PE
Zombie NIR
monensin
RORγt APC
Aurora spectral cytometer
Acetate
Af647
Antibodies
Biotin
Cd4 t cells
Cells
Cy5 5
Fitc
Flow cytometry
Il 17a
Intracellular
Ionomycin
Lymphocytes
Monensin
Myristate
Rorγt
Streptavidin
T cell
Corresponding Organization :
Other organizations : Icahn School of Medicine at Mount Sinai
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Variable analysis
independent variables
- Stimulation with 5 ng/mL phorbal 12-myristate 13-acetate and 500 ng/mL ionomycin in the presence of monensin for 3.5 h
- Intracellular IL-17A expression
- Expression of transcription factors FoxP3 and RORγt
- T cell Vβ chain repertoire
- Unstimulated cells for analysis of FoxP3 and RORγt expression
- Positive control: Stimulation with phorbal 12-myristate 13-acetate and ionomycin to induce intracellular IL-17A expression
- Negative control: Unstimulated cells for analysis of FoxP3 and RORγt expression
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