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Human peripheral blood mononuclear cells (PBMCs) were isolated with the CD4 + T Cell Isolation Kit, followed by a CD25 /CD45RO depletion. Afterwards, cells were either left unstimulated (left images) or stimulated with CD3 and CD28 antibodies as well as with human IL-4 and human IL-2 for three days. Cells were then polarized with human IL-4 and human IL-2 and restimulated with PMA and ionomycin. After two hours brefeldin A was added. Cells were fixed, permeabilized, and intracellularly stained with Anti-IL-9 antibodies as well as with CD4 and CD154 antibodies. CD4 + cells were pre-gated for the analysis. Cells were then analyzed by flow cytometry using the MACSQuant ® Analyzer. Cell debris were excluded from the analysis based on scatter signals. |
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