Bone Marrow Macrophage Polarization

Bone marrow (BM) cells were flushed from femur and tibia of Adrb2^+/+ and Adrb2^−/− mice and cultured using DMEM supplemented with 20% (v/v) FBS, 1% (v/v) P/S, 20 ng/ml mouse recombined murine M-CSF (Proprotech, USA) for 7 days (17 (link)). The purity of BM-macrophage cultures was confirmed by FACS using CD11b (1:100, BioLegend, San Diego, CA, USA) and F4/80 (1:100, BioLegend, San Diego, CA, USA) antibodies. Seven days later, BM-derived macrophages were cultured in a DMEM medium (Invitrogen, Carlsbad, CA, USA) for 24 h to remove the residual M-CSF. M0 macrophages (non-differentiated macrophages) were then stimulated with recombinant IL-4 (rIL4) (20 ng/ml, Proprotech, USA) with or without mTORC1 inhibitor rapamycin (100 ng/ml, MCE, USA) for 24 h. After that, the rIL-4 was removed and the cells were washed to remove the remaining rIL-4 and collected for further analysis. For measuring IL-4, the cells were washed and the DMEM medium (Invitrogen, Carlsbad, CA, USA) was added for another 24 h.