Description
The ex vivo addition of BD GolgiPlug™, a protein transport inhibitor containing brefeldin A, to in vitro- or in vivo-stimulated lymphoid cells blocks their intracellular protein transport processes. This results in the accumulation of cytokines and/or proteins in the Golgi complex. This increased accumulation of cytokines in the cell enhances the detectability of cytokine-producing cells with immunofluorescent staining and flow cytometric analysis.
Brand:BD GolgiPlug™
Regulatory Status:RUO
RRID:AB_2869014
Application:Intracellular staining (flow cytometry) (Routinely Tested)
Preparation And Storage
Store undiluted at 4°C.
Recommended Assay Procedures
Stimulation of cells: Various in vitro methods have been reported for stimulated 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 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).
Procedure for using BD GolgiPlug™: Add 1 µl of BD GolgiPlug™ for every 1 ml of cell culture (e.g., ~10^6 cells/mL) and mix thoroughly. Treatment of stimulated cells for 4 to 6 hours with BD GolgiPlug™ significantly increases the ability to detect cytokine-producing cells by immunofluorescent staining. It is recommended that BD GolgiPlug™ not be kept in cell culture for longer than 12 hours.
As an alternative to BD GolgiPlug™, investigators may wish to consider using BD GolgiStop™, a protein transport inhibitor containing monensin (Cat. No. 554724). BD GolgiPlug™ and BD GolgiStop™ have been found to have differential effects on intracellular cytokine staining that is time, activator and cytokine dependent. These factors need to be considered when carrying out intracellular cytokine staining.
