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Splenocytes from a KLH-immunized mouse were restimulated in vitro with or without KLH, CD28, and CD40 antibodies for six hours. Cells were harvested, stained with CD4-FITC and CD154-PE, or CD154-Biotin and Anti-Biotin-PE, and analyzed by flow cytometry. A lymphocyte gate based on forward and side scatter properties was activated. Dead cells and B cells were excluded according to PI and CD45R/B220-PerCP™ staining.
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