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T cells were isolated using the CD4+
T Cell Isolation Kit II from mouse spleen cells and stimulated with CD3ε pure – functional grade and CD28 pure – functional grade antibodies in the presence of recombinant Mouse IL-2 and Mouse IL-4 and Anti-IFN-γ pure – functional grade antibody for four days. Then cells were harvested, washed, and incubated with or without (left image) PMA/Ionomycin for six hours. After two hours, brefeldin A was added. The cells were harvested, fixed, permeabilized, and intracellularly stained with Anti-IL-3, CD4-FITC and CD154-APC or CD154-PE. Cells were analyzed by flow cytometry using the MACSQuant®
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