Hello Mats Urban ,

Here is an answer from BD ( https://www.bdbiosciences.com/content/dam/bdb/marketing-documents/Cytokines_FAQ.pdf )

Q. What is the difference between the BD GolgiStop™ and BD GolgiPlug™ protein transport inhibitors?

A. The major difference between these two inhibitors is where they work within a cell. BD GolgiStop (monensin) works by accumulation of protein at the endoplasmic reticulum (ER) stage, while BD GolgiPlug (brefeldin) works with accumulation of protein at the golgi complex. The use of these inhibitors leads to an enhanced ability to detect cytokine-producing cells by intracellular flow cytometry.

Never worked with bone marrow cells before, only with PBMC. For intracellular cytokine staining, we used PMA/ Ionomycin/ Brefeldin-A (first paper) or PMA/ Ionomycin/ Monensin (second paper) and we are very happy with the results. PMA is a very potent cell activator (triggers maximal activation so you can see the "maximal" secretory potential of your cells).You can find the concentrations used by us in these articles.Article Variability of ex-vivo stimulated T-cells secretory profile ...

Article Treatment With Cladribine Selects IFNγ+IL17+ T Cells in RRMS...

About the concentrations, here's a snippet from the first paper:

Several concentrations of PMA/ION are report -ed in the literature for PBMC activation, rangingfrom 1 to 100ng/mL PMA (32-39) and from 0.4to 1.4μM for ION (that is roughly 300-1000ng/mL) (32-36,38), depending on protocol require-ments and personal preferences. While the exactPMA concentration values seem to be chosen ar-bitrarily (e.g. 1, 2.5, 5, 20, 25, 50 or 100ng/mL),most studies recommend that ION be added at aconcentration of 1μM, that is 710ng/mL. Also,the incubation time may vary between protocols,depending on the desired downstream applica -tion of the activated cells. Short-term activationrequires a minimum of 4-6h (32-38) and usual -ly employs higher concentrations of PMA/ION,while longer activation times may reach up to24-72h (33,37). BD makes recommendations on the optimal concentration of each ingredient forcellular activation purposes: PMA 5ng/mL, ion-omycin 500ng/mL and BD GolgiPlug 1μL/mLof cell culture.

About the type of protein transport inhibitor, there are several sources on-line saying that monensin is more toxic than brefeldin and also monensin does not completely block TNF secretion.

I hope this helps you at least to get an idea about this technique.

Good luck!