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Mouse spleen cells were incubated with or without PMA/ionomycin for 6 hours. After 2 hours, brefeldin A was added. Cells were then fixed, permeabilized, and intracellularly stained with Anti-GM‑CSF antibodies and CD4‑FITC and CD154-APC or CD154-PE and CD45R (B220)‑PerCP and analyzed by flow cytometry using the MACSQuant®
Analyzer. The unstimulated controls can be seen in the left image. Gating was performed according to CD4 expression and side scatter properties of the cells. B cells and cell debris were excluded from the analysis in a fluorescence 3 versus fluorescence 4 dot plot.
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