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T cells were enriched (CD4+
T Cell Isolation Kit II, mouse) from spleen cells of a BALB/c mouse. Cells were either left unstimulated (left image) or stimulated with plate-bound CD3 and soluble CD28 antibodies in the presence of recombinant TGF-beta, recombinant IL-4, recombinant IL-2, and neutralizing Anti-IL-6 antibody, Anti- IFN-γ antibody and Anti-IL-12 antibody. On day 5, brefeldin A was added for 4 hours. The cells were harvested, fixed, permeabilized, and intracellularly stained with Anti-IL-9 antibodies, as well as with CD4-FITC. Cells were analyzed by flow cytometry using the MACSQuant®
Analyzer. Gating was performed according to forward scatter and side scatter properties of the cells.
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