3t3 l1 preadipocytes

3T3-L1 preadipocytes were obtained from American Type Culture Collection (ATCC Manassas, VA, USA) and cultured in a humidified atmosphere with 5% CO₂ at 37 °C. The cells were differentiated into adipocytes as previously reported [14 (link)]. Briefly, 3T3-L1 preadipocytes were cultured in Dulbecco’s modified Eagle’s medium (DMEM) containing 10% bovine calf serum (Gibco life technologies, Carlsbad, CA, USA) and 1% antibiotic–antimycotic solution (Gibco life technologies). For the differentiation assay, preadipocytes were seeded in six-well plates and cultured until two days post-confluence. After the 3T3-L1 cells became confluent, the medium was replaced with DMEM consisting of 10% of fetal calf serum (FBS; Gibco-BRL), 1% of antibiotic–antimycotic solution, 10 μg/mL bovine insulin, 1 μM dexamethasone (Sigma-Aldrich, St. Louis, MO, USA), and 0.5 mM isobutylmethylxanthine (Sigma-Aldrich) with or without filbertone (Sigma-Aldrich, St. Louis, MO, USA) and SQ22,536 (an adenylate cyclases (ADCY) inhibitor, Sigma-Aldrich, St. Louis, MO, USA). The cells were then maintained in DMEM containing 10% of FBS with 10 μg/mL insulin for another two days, followed by culturing with DMEM containing 10% of FBS for an additional four days with or without filbertone and SQ22,536.

3T3-L1 preadipocytes were obtained from the Procell Life Science&Technology Co., Ltd. (Wuhan, Hubei, China). After resuscitation, 3T3-L1 preadipocytes were cultured in Dulbecco’s modified Eagle’s medium (DMEM; #11965-092, Gibco, Carlsbad, CA, USA) supplemented with 10% calf serum (CS; #16010-159, Gibco, Carlsbad, CA, USA) and 1% penicillin-streptomycin (#10378-016, Gibco, Carlsbad, CA, USA) at 37°C in a humidified incubator containing 5% CO₂. The differentiation process of the cells was performed as previously described [27 (link)]. Briefly, two days after cell confluency, cells were stimulated with DMEM containing 10% (v/v) foetal bovine serum (FBS; #10100-147, Gibco, Carlsbad, CA, USA), 10 μg/ml insulin (#HY-P1156, MedChemExpress, Shanghai, China), 0.5 mM isobutylmethylxanthine (#HY-12318, MedChemExpress, Shanghai, China) and 1 μM dexamethasone (#HY-14648, MedChemExpress, Shanghai, China) for 48 h to induce differentiation. Then, the culture medium was replaced with DMEM supplemented with 10% FBS and 10 μg/ ml insulin. After 48 h, the medium was replaced with DMEM containing 10% FBS without insulin and changed every 2 days. Cells were used at 9 to 10 days after induction of differentiation, at which time 90% of cells exhibited an adipocyte phenotype.

The 3T3-L1 mouse embryo fibroblasts (termed 3T3-L1 preadipocytes) were purchased from American Type Culture Collection (Manassas, VA, USA). 3T3-L1 preadipocytes were maintained in Dulbecco’s Modified Eagle’s Medium (DMEM, Gibco, Carlsbad, CA, USA) supplemented with 10% fetal bovine serum (ExCell, Shanghai, CHN) and 1% antibiotic (10,000 U/mL penicillin and 10,000 U/mL streptomycin, PanEra, Guangzhou, CHN) at 37 °C in 5% CO₂ in a humidified incubator. After becoming completely confluent, 3T3-L1 preadipocytes were stimulated to differentiate in the above growth medium containing 10 μg/mL insulin, 0.5 mmol/L 3-isobutyl-1-methyl-xanthine (IBMX) and 1 μmol/L dexamethasone (all from Sigma, St. Louis, MO, USA) for 2 days, followed by exposing to the growth medium only containing 10 μg/mL insulin for 2 more days. At day 4, cells were maintained in growth medium again for 4 to 6 days until more than 85% of them were filled with lipid droplets. Mature 3T3-L1 adipocytes were identified by Oil Red O staining (Fig. 1).Fig. 1

Differentiation of 3T3-LI adipocytes. 3T3-L1 adipocytes at day 4 (a), 6 (b) and 8 (c) after differentiation. Mature 3T3-L1 adipocytes were stained by Oil Red O (d)