Recombinant murine tnf α

Murine 3T3-L1 preadipocytes (obtained from Dr. Howard Green at Harvard University) were grown to confluence and differentiated into lipid-laden mature adipocytes as indicated in [27 (link)]. The development of lipid droplets following differentiation was used to consistently confirm the identity of the 3T3-L1 adipocyte cell line. Prior to treatment with murine TNFα, the adipocytes were serum-starved overnight up to 24 h by changing the 10% serum medium to Dulbecco’s Modified Eagle’s Media (DMEM) (Sigma–Aldrich, St. Louis, MO, USA) containing 1% calf serum. Recombinant murine TNFα was purchased from Thermo Fisher (Waltham, MA, USA; Cat #: PMC3013). All cell culture experiments were performed in Corning^® Costar^® tissue culture (TC)-treated 10 cm or multiwell plates.

Mouse glucagonoma αTC1.6 cells were purchased from the American Type Culture Collection (ATCC). Cells were maintained in Dulbecco's Modified Eagle Medium (DMEM—Sigma-Aldrich, Saint Louis, MO, USA) containing 10% fetal bovine serum (FBS), 2 mM L-glutamine, 0.15% 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) 15 mM, 1% non-essential amino acids (NEAA), 0.02% bovine serum albumins (BSA, Sigma-Aldrich), 25 mM d-glucose (Sigma-Aldrich), 100 U/ml penicillin, and 100 μg/ml streptomycin, at 37°C, with 5%-CO₂ humidified incubator. Mouse insulinoma βTC1 cells were also from ATCC; cells were cultured in DMEM with 25 mM glucose (Sigma Aldrich), supplemented with 2 mM L-Glutamine, 15% horse serum (HS), 2.5% FBS, 1% penicillin/streptomycin, in 95% humidified air, with 5% CO₂ at 37°C. Cells were passaged once a week after trypsinization and replaced with new medium twice weekly. Both cell lines were treated with a cytokine cocktail (recombinant murine IL-1β, specific activity 0.1 U/ml, Peprotech, London, UK, UE; recombinant murine IFN-γ, specific activity 25 U/ml, Peprotech; recombinant murine TNF-α, specific activity 25 U/ml, Peprotech), as previously described (22 (link)).