IL-4 (860A13C4), Mouse Monoclonal Antibody - UNCONJ - FAQs

How does monensin help in intracellular staining of some secreted proteins?

Monensin is an Na+ ionophore. Treatment of cells with monensin disrupts intracellular Na+ and H+ gradients, and thereby inhibits Golgi transport and secretion. In monensin-treated cells, nascently synthesized proteins are retained and concentrated within the cell. This increased protein concentration facilitates detection of specific proteins by the intracellular staining technique.

Treating cells with monensin is especially useful when monitoring the efficacy of stimulation protocols which induce rapid or transient increases in protein synthesis. An example of such a stimulation protocol is the treatment of cells with phorbol myristate acetate (PMA) plus Ca2+ ionophore A23187. This stimulation protocol rapidly up-regulates IL-4 synthesis. We have observed that when isolated human PBMC's are treated concurrently with PMA/A23187 plus monensin, significant levels of IL-4 production are detected within a subpopulation by the intracellular staining technique. However, when the stimulation is performed with PMA/A23187 alone, IL-4 production cannot be detected by this technique.

We have evaluated the effect of the timing of monensin addition on our ability to detect IL-4, plus several other cytokines including IL-2 and INF-gamma. Our experimental results indicate that monensin addition timing should be optimized for the particular stimulation protocol and cytokine under investigation. However, in all cases examined, we find that adding monensin at the beginning of the stimulation protocol allows us to detect higher levels of cytokine than when we omit monensin from the experiment.

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