Macrophage Polarization and NF-κB Signaling

Mouse macrophages were derived as described elsewhere [14 (link)]. Briefly, bone marrows were collected from the femur and tibia of C57BL/6 mice. The marrow cells were cultured in DMEM medium supplemented with 10% FBS, 1% penicillin-streptomycin (all from Invitrogen), and 20 ng/ml recombinant mouse M-CSF (PeproTech, Rocky Hill, USA) for 5 days to differentiate into macrophages. For analysis of macrophage polarization, cells were cultured with 20 ng/ml of mouse recombinant IL-4 or 20 ng/ml of mouse recombinant IL-10 (PeproTech) for 24 h, together with or without 20 μmol/L of embelin. For macrophage activation and NF-κB signaling assays, cells were pretreated with embelin for 1 h, and then stimulated with LPS (500 ng/ml, Sigma-Aldrich) for indicated durations.