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2 protocols using tae beads anti cd2 cd3 cd28

1

Multiparameter Immunophenotyping of T Cells

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Alexa488-conjugated anti-GATA3, Alexa647-conjugated anti-CXCR5, APC-Cy7-conjugated anti-CD4, BUV395-conjugated anti-IFNγ, BV711-conjugated anti-IL-2, Pe-Cy7-conjugated anti-CD25, PE-conjugated anti-CCR6 and anti-mouse IgG1, and PerCpCy5.5-conjugated anti-CD127 and anti-Tbet were purchased from Becton Dickinson. Alexa488-conjugated anti-IL-10, eFluor660-conjugated anti-IL-21, FITC-conjugated anti-CD45RA, PE-conjugated IL-22, Pe-Cy7-conjugated anti-IL-4 and mouse IgG1 were purchased from eBiosciences. APC-Cy7-conjugated anti-IL-17A, BV421-conjugated anti-CXCR3, and BV605-conjugated anti-TNFα was purchased from Biolegend. FITC-conjugated anti-CCR7 and recombinant human IL-12 was purchased from R&D Systems. Anti-DOCK8 mAb was purchased from Santa Cruz Biotechnology. Recombinant human TGFβ, IL-1β, IL-6, IL-21 and IL-23 were from Peprotech. Prostaglandin E2, PMA, calcium ionophore (ionomycin), Brefeldin A, and saponin were purchased from Sigma-Aldrich and recombinant human IL-4 was provided by Dr Rene de Waal Malefyt (DNAX Research Institute, Palo Alto, CA). T cell activation and expansion (TAE) beads (anti-CD2/CD3/CD28) were purchased from Miltenyi Biotec and CFSE was purchased from Invitrogen.
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2

Isolation and Cytokine Profiling of Naive and Memory CD4+ T Cells

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Naïve and memory CD4+ T cells were isolated by excluding Tregs (CD25hiCD127lo) and then sorting CD45RA+CCR7+ cells and CD45RACCR7+/− cells, respectively, ensuring >98% purity of the recovered populations. Sorted naïve and memory CD4+ T cells were cultured in 96-well round-bottom plates (40 × 103 cells/well) with TAE beads (anti-CD2/CD3/CD28; Miltenyi Biotech). After 5 d, supernatants were harvested to measure secretion of IL-4, IL-5, IL-13, IL-17A, IL-17F, IFN-γ, and TNF-α using cytometric bead arrays (Becton Dickinson). IL-22 secretion was measured by ELISA (PeproTech). Intracellular cytokine expression was determined following re-stimulation of cells with PMA and ionomycin with addition of Brefeldin A (10 μg/ml) after 2 h.
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