4.4. Adipocyte Cell Culture and Differentiation

3T3-L1 pre-adipocytes were cultured in GM2. Differentiation was induced 2 days post-confluence by adding GM2 containing 0.5 mM 3-isobutyl-1-methylxanthine (IBMX), 0.25 μM dexamethasone, 2 μM Rosiglitazone (Sigma- Aldrich, St. Louis, MO, USA) and 1 μg/mL insulin (Novo Nordisk, Bagsværd Denmark). After 2 days of incubation, medium was replaced with GM2 containing 1 μg/mL insulin. Two days later, medium was replaced by GM2 and incubated for another 7 days with the different treatments (Replacing it every 2 days). MTT cell proliferation, 7-Amino Actinomycin D (7-AAD) cell viability, Dihydrorhodamine (DHR) and Mito Tracker flow cytometry assays were performed. Cells were stained to evaluate intracellular triglyceride (TG) accumulation, supernatants were collected to evaluate adipokine concentration, RNA was purified to evaluate functional gene transcription and protein was extracted for western blot analysis.