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.
Suggested Companion Products
Protein Transport Inhibitor (Containing Monensin) RUO 0.7mLCat No: 554724
BD Cytofix/Cytoperm Plus Kit (with BD GolgiPlug) RUO 250TestsCat No: 555028
BD Cytofix/Cytoperm Plus Kit (with BD GolgiStop) RUO 250TestsCat No: 554715
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Preparation and Storage
Store undiluted at 4°C.
Product Notices
- Please refer to www.bdbiosciences.com/pharmingen/protocols for technical protocols.
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.
