Recombinant mouse il 10

Mouse SVF cells were grown in DMEM/F12 media (Hyclone) supplemented with 10% FBS. For beige or brown adipocyte differentiation, confluent cells (D0) were changed to medium A containing 0.5 mM IBMX, 1 µM dexamethasone, 125 nM indomethacin, 850 nM insulin, 1 nM T3 and 1 µM rosiglitazone (all from Sigma, Saint Louis, MO) for 2 days, then changed to medium B containing 850 nM insulin, 1 nM T3 and 1 µM rosiglitazone for another 5 days. H1.2 knockout or Il10rα overexpression was achieved by infecting differentiated SVF cells which were isolated from H1.2^flox/flox mice with adenovirus-packed Cre-GFP or AAV packed Il10rα (Obio Tech.; MOI = 100) at D2. At D7, differentiated beige adipocytes were incubated with or without 10 µM isoproterenol (Sigma) for 4 h. For Il10 stimulation, 50 ng/ml or 100 ng/ml recombinant mouse Il10 (Peprotech, Rocky Hill, NJ) was added to differentiated beige adipocytes for 12 h. For Oil Red O staining, differentiated cells were fixed with 4% formaldehyde, and stained with 0.5% Oil Red O (Sigma) dissolved in propylene glycol at D7^{15 (link),17 (link),31 (link)}.

Bone marrow-derived macrophages (BMDMs) were prepared as described previously24 (link). In brief, bone marrow cells were grown in L-cell-conditioned IMDM medium supplemented with 10% FBS, 1% non-essential amino acid and 1% penicillin-streptomycin for 5 days to differentiate into macrophages. On day 5 BMDMs were seeded in 6-well cell culture plates. On the next day BMDMs were stimulated with LPS (Invivogen- LPS-SM) (20 ng/ml) or PAM3CSK4 (1 μg/ml) for the indicated hours of time followed by 5 mM ATP or 20 μM nigericin for the last 30 minutes. In some experiments, BMDMs were pretreated for 30 minutes with mouse recombinant IL-10 (50 ng/mL- Peprotech) or anti-mouse IL-10R mAb (1 μg/mL- BioxCell) before LPS/PAM3CSK4 stimulation.
In supernatant transfer experiments, supernatants from control (0 h sups), 4 h LPS treated (4 h sups) and 24 h LPS treated (24 h sups) BMDMs were transferred to fresh unstimulated BMDMs. These BMDMs were then stimulated with 20 ng/ml LPS for 4 h followed by ATP for the last 30 minutes. Caspase-1 activation was determined in the cell lysates and IL-1β and IL-18 production were determined in the supernatants by ELISA.

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.