The most critical step for detection of intracellular accumulations of cytokines by intracellular staining is the isolation and activation of a cell population to induce production of the cytokine of interest. Without properly activated cytokine-producing cells, cultured in the presence of a protein transport inhibitor, the ability to detect intracellular accumulations of cytokines in most cases is severely compromised. Various in vitro methods have been reported for stimulating cells to produce cytokines. Polyclonal activators have been particularly useful for inducing cytokine-producing cells. These activators include the following: concanavalin A, lipopolysaccharide, phorbol esters plus calcium ionophore or ionomycin, phytohaemaglutinin, staphylococcus enterotoxin B, and monoclonal antibodies directed against subunits of the TCR/CD3 complex (with or without antibodies directed against costimulatory receptors, such as CD28).
The Leukocyte Activation Cocktail, with BD GolgiPlug™ is a ready -to-use polyclonal cell activation mixture containing the phorbol ester, PMA (Phorbol 12-Myristate 13-Acetate), a calcium ionophore (Ionomycin) and the protein transport inhibitor BD GolgiPlug™ (Brefeldin A). This mixture may be utilized to elicit a primary cytokine response from T cells. Stimulation of cells using this cocktail results in cytokine production that is localized in the rough endoplasmic reticulum of cytokine -producing cells. This localization of cytokines is caused by the protein transport inhibitor component, Brefeldin A which blocks intracellular protein transport processes. The increased accumulation of cytokine protein levels in cells thus enhances detectability of cytokine-producing cells by immunofluorescent staining and flow cytometric analysis. The contents of each 100 µL vial is sufficient for treating up to 50 mL of cell cultures (at ~10^6 cells/mL); therefore one can stimulate ≥ 5 × 10^7 cells.