Cell culture and differentiation of 3T3-L1 into mature adipocytes

3T3-L1 (CRL-173) preadipocytes were optained from the American Type Culture Collection (ATCC, Manassas, VA). Preadipocyte expansion medium was prepared 90% DMEM supplemented with 10% FBS, 1% L-Glutamine and 1% Penicillin+streptomycin antibiotics. The cells were cultured in 25 cm² flask containing a preadipocyte expansion medium. The cells were grown in an incubator (Nuaire, NU-5800, USA) with a humidified air at 37°C containing 5% CO². The culture medium was altered every two-three day in cabinet (LabGard NU-540, Nuaire, US) and cells observed using an inverted microscope (Leica, Dmil Led Fluo, Thermo Fisher, Germany).

The differentiation of 3T3-L1 preadipocytes into mature adipocytes were cultured and maintained [19]. Differentiation medium was prepared 90% DMEM supplemented with 10% FBS, 1% L-Glutamine, 1% Penicillin+streptomycin antibiotics-containing DEX (1μM), Insulin (1 μg/mL), and IBMX (0.5 mM). First, 3T3-L1 cells were seeded preadipocyte expansion medium at 8x10⁴ in 6-well plate containing 1 mL medium until reached confluence. After 48 h for the induction of differentiation 3T3-L1 preadipocyte were cultured with differentiation medium. After 72 h the induction of differentiation, the differentiation medium was removed and added by adipocyte maintenance DMEM supplemented with 10% FBS and insulin (1μg/mL) for another 48 h. After 5 days the induction of differentiation, the fresh adipocyte maintenance medium was replaced every two days until 14 days. The visible of lipid accumulation was monitored with an inverted microscope (Leica, Dmil Led Fluo, Thermo Fisher, Germany) [20, 21]. The mature 3T3-L1 adipocytes were used for further analysis.