For cytokine staining, CD4+ T cells activated under various polarizing conditions were restimulated for 4 h with phorbol 12-myristate 13-acetate (50 ng/ml) and ionomycin (550 ng/ml; Sigma-Aldrich) in the presence of GolgiStop (BD PharMingen). Cells were then fixed and permeablized with Foxp3 staining buffer set or Cytofix/Cytoperm solution to preserve the GFP signal, and then stained with fluorochrome conjugated anti–IL-9 (RM9A4), anti–IL-4 (11B11), and anti-CD4 (GK1.5) antibodies (all from Biolegend) according to the manufacturer’s instructions. All samples were acquired using LSRII, and data were analyzed with FlowJo v10 software (Xiao et al., 2016 (link)).
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Cytokine Profiling of Activated CD4+ T Cells
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Corresponding Organization : Cornell University
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Variable analysis
independent variables
- Various polarizing conditions used to activate CD4+ T cells
- Expression of IL-9, IL-4, and CD4 in the activated CD4+ T cells
- Phorbol 12-myristate 13-acetate (50 ng/ml) and ionomycin (550 ng/ml) used to restimulate the CD4+ T cells
- GolgiStop used to preserve the intracellular cytokine signals
- Fixation and permeabilization of cells using Foxp3 staining buffer set or Cytoffix/Cytoperm solution to preserve the GFP signal
- Fluorochrome-conjugated antibodies used to stain for IL-9, IL-4, and CD4
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