Indirect Co-culture of Adipocytes and Macrophages

Mouse preadipocytes (3T3-L1) and macrophages (RAW264.7) were obtained from American Type Culture Collection (ATCC) (Manassas, VA, USA), cultured, and induced to differentiate in a growth medium as previously reported [17 (link)]. For indirect co-culture of 3T3L1 and RAW264.7 cells, media from RAW264.7 cells were collected and sterile filtered (0.2 μm). The differentiation of 3T3-L1 cells was induced using MaCM supplemented with a differentiation cocktail (1% PS, 10% bovine calf serum (BCS), 0.5 mM 3-isobutyl1-methylxanthine, 1μM dexamethasone, and 1μg/mL insulin) for 2 days. The medium was then changed to MaCM with 10% BCS and insulin (1μg/mL) and was replaced every 2 days through day 8. At least 3 independent experiments were performed for each condition. Human primary preadipocyte cells from hSAT and hVAT (#PT5001 and #PT5005, Lonza, Walkersville, MD, USA) were processed and differentiated according to the supplier’s instructions in 2 independent experiments.