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Stimulation reagents, phorbol 12-myristate 13-acetate (PMA), ionomycin, brefeldin A, and monensin are useful to activate transcription factors for intracellular signaling and production of cytokines of many different immune cell types. Brefeldin A solution is required for intracellular retention of signaling proteins and cytokines. Brefeldin A is an inhibitor of intracellular protein transport. Incubation of cells in culture with brefeldin A leads to blockade of protein transport to the Golgi complex (GC) and accumulation of proteins in the endoplasmic reticulum (ER). Addition of brefeldin A during the last hours of in vitro activation of cells results in enhanced detection of intracellular cytokines. If performing an immunoassay on secreted protein (e.g., ELISA, western or multiplex protein detection), cells do not require treatment with brefeldin A.
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For Research Use Only. Not for use in diagnostic procedures.