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Mouse spleen 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-5-PE, CD4-FITC, and CD154-APC. Cells were analyzed by flow cytometry using the MACSQuant® Analyzer. Gating was performed according to the CD4 expression and side scatter properties of the cells.
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