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Naive mouse T cells were isolated using the CD4+
T Cell Isolation Kit and stimulated with CD3 and CD28 pure – functional grade antibodies in the presence of recombinant mouse IL-2 and IL-4 and Anti-IFN-γ antibody pure – functional grade for four days. Afterwards, cells were incubated with or without PMA/Ionomycin for 6 hours. After 2 hours, brefeldin A was added. The cells were harvested, fixed, permeabilized, and intracellularly stained with Anti-IL-4-PE, CD4-FITC, and CD154-APC (A), or Anti-IL-4-APC, CD4-FITC, and CD154-PE (B). 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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