Stimulation of Cells: 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, staphlylococcus, entertoxin 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 GolgiStop™: Add 4 µl of BD GolgiStop™ for every 6 mL of cell culture (e.g., ~10^6 cells/mL) and mix thoroughly. Treatment of stimulated cells for 4 to 6 hours with BD GolgiStop™ significantly increases the ability to detect cytokine-producing cells by immunofluorescent staining. It is recommended that BD GolgiStop™ not be kept in cell culture for longer than 12 hours.
As an alternative to BD GolgiStop™, investigators may wish to use BD GolgiPlug™ , a protein transport inhibitor containing brefeldin A (Cat. No. 555029). BD GolgiStop™ and BD GolgiPlug™ have been found to have differential effects on intracellular cytokine staining that is time, activator and cytokine dependent. These factors must be considered when carrying out intracellular staining.
Danger: BD GolgiStop™ Protein Transport Inhibitor, containing monensin (component 51-2092KZ) contains 99.61% ethanol (w/w) and 0.26% monensin, mononatriumsalz (w/w).
Highly flammable liquid and vapor.
Causes serious eye irritation.
Harmful if swallowed.
Keep away from heat/sparks/open flames/hot surfaces. No smoking.
Wear protective gloves / eye protection.
Wear protective clothing.
IF ON SKIN (or hair): Remove / Take off immediately all contaminated clothing. Rinse skin with water / shower.
IF IN EYES: Rinse cautiously with water for several minutes. Remove contact lenses, if present and easy to do. Continue rinsing.
IF SWALLOWED: Call a POISON CENTRE/doctor if you feel unwell. Rinse mouth.
Dispose of contents / container in accordance with local / regional / national / international regulations.Keep container tightly closed.