Secretion stop

Cell source Stimulation Secretion stop Fixation Permeabilisation Staining Protocol

 

Up


After stimulation, induction of the target proteins they are quickly moved to their site or secreted. To detect for instance cytokines intracellular it is useful to add a secretion inhibitor to your cells. Of course this is only possible (and useful) when the cells have been cultured and/or activated in vitro. Sometimes it is interesting to look at cytokine profiles from cells directly from the tissue. The levels of cytokine expressed will be very low and most of the time the production will be just above or below the detection level. 
Most protocols will stimulate the cells in vitro and add one of the two secretion inhibitors monensin or brefeldin A.

Monensin.

The secretion inhibitor monensin is a Na+ ionophore. Which block glycoprotein secretion.
Monensin isn't only more toxic than brefeldin it has been reports that it also induces the production of IL-1 and TNFa by monocytes within 30 minutes. So experiments with monocytes, macrophages, monocyte derived dendritic cells and any other monocyte containing population, like PBMC, can't be done with the use of monensin. 

Brefeldin A.

This second inhibitor is brefeldin A, a carboxylated ionophore, that blocks reversibly the transport from the Golgi to the endoplasmatic reticulum. It is isolated from Penicillium brefeldianum. The concentration brefeldin is used at is 10 g/ml. The advantage of using this inhibitor is the fact that you can add it to your culture for a maximum of 30 hours. 

Both chemicals should be dissolved in DMSO. They can be aliquoted and stored at -20C. Before bringing them to the concentration you will add to your culture dilute the stock in PBS. Some laboratories dilute in 70% ethanol first. This doesn't harm the culture as long as the volume percentage of alcohol is minimal in relation to the final volume.
Keep in mind that you do not only add the inhibitor to your stimulated cultures but also to the non-stimulated (control) cultures. And add a minimum of volume to your cultures (high concentration inhibitor), not to dilute your stimulus too much. Both chemicals are already influencing the viability of the culture after 8-10 hours. Brefeldin is the least toxic from both. Some laboratories use one inhibitor for one type of cytokine and the other for other cytokines. In our experience there is not much of a difference between the two inhibitors.
Of course every cell type (see remark concerning monocytes) and protein will behave different in the presence of these inhibitors and testing is the only solution.

 

Although we did our best we can not be held responsible for any mistakes or typing errors.
 Suggestions, literature and protocols are welcome at icstaining@schuitemaker.info. Copyright 2000 - 2005.
Page was last updated: 05-03-2005