I used the combination of PMA (50 ng/ml) and Ionomycin (1 microg/ml) to stimulate peripheral mononuclear cells. I'm not sure the exact suitable concentration for murine peritoneal macrophage, but you can start with the above concentration, first.

We normally use 1 micromolar concentration of PMA for 1-4 hours.

Concentration strongly depends of your aim.

please analyse previous conversation on RG:

https://www.researchgate.net/post/how_the_THP-1_cells_become_adherent_with_PMA_what_is_the_mechanism_and_how_long_they_still_adherent_on_the_surfece_in_incubation_culture_media

and this

https://www.researchgate.net/post/Why_THP-1_cells_wont_attach_after_PMA_stimulation

Thank you very much for the answers. I have tried 20ng/mL PMA and 1.5 microM but the murine peritoneal macrophages get up and seem died on ly with both stimulation. The aim is study protein expression, so I need enough time to this and I do not know whether a stimulation of 5 hours is enough. Best regards !

It is important to optimize used concentration since overstimulation leads to the suicide of macrophages. F.e. 20 ng/ml corresponds to 20 ug/L and this concentration of PMA induces very strong ROS production in macrophages. The viability of the rat resident peritoneal macrophages 24 hr after stimulation with such a dose is around 60 % of control. The effect could be partially abrogated by DMSO with conc. > 0.1 % we used as a solvent for PMA.

Thank you very much for your answer!!

Very interesting,

Best,

Javier

Hi Javier,

You are welcome. At the same time, you asked about it a long time ago. Probably you have some comments on your own question now. Please share it if yes.

Our results obtained on resident (not elicited) peritoneal cells population (Mfs, Mast cells, Ly) isolated from Wistar rats. I believe peritoneal cells from mice could demonstrate different responses and the presence of other than macrophages cell as well could account to the results.