3T3-L1 Adipocyte Differentiation Protocol

Pre-adipocytes were seeded in tissue culture plates coated with 0.1% gelatin (Millipore, Burlington, MA, USA) and grown until confluency at 37°C in a humidified atmosphere with 5% CO2 in complete medium (CM) containing 4.5 g/L glucose DMEM (ThermoFisher Scientific, Waltham, MA, USA), 10% foetal bovine serum, 100 IU/ml penicillin and 100 μg/ml streptomycin. Differentiation of 3T3-L1 cells was initiated two days after reaching confluency (day 0) using CM supplemented with 500 μM 3-isobutyl-1-methylxanthine (IBMX, ThermoFisher Scientific), 1 μM dexamethasone, 1.7 μM human insulin (Actrapid, Novo Nordisk, Bagsværd, Denmark) and 1 μM rosiglitazone (ThermoFisher Scientific). On day 3, cells were switched to CM containing 0.5 μM insulin for 48 hours (day 5). To complete differentiation, cells were cultured in CM with 2% FBS until day 7 and from here onwards maintained on CM containing 1 g/L glucose DMEM (ThermoFisher Scientific) and 2% FBS for two days. Subcutaneous pre-adipocytes were differentiated using a similar protocol until day 5 and from then onwards maintained on 10% FBS medium as previously described [34 (link)]. Mature adipocytes were treated with recombinant murine IL-27 (Biolegend, San Diego, CA, USA) or vehicle control (PBS supplemented with 1% BSA) as indicated.