3t3 l1 preadipocytes

3T3-L1 preadipocytes were purchased from Sigma (Cat. No. 86052701) and maintained in Dulbecco′s modified Eagle′s medium (DMEM, Sigma, Cat. No. D5030) with 10% bovine calf serum (BCS, Sigma, Cat. No. 12133C) and avoided complete confluence before initiating differentiation [3 (link)]. For adipogenesis, preadipocytes were cultured in 24-well plates (10⁴ cells per well) and differentiation was induced for 48 h by DMEM supplemented by 10% BCS, 0.05 mM 3-isobutyl-1-methylxanthine (Sigma, Cat. No. I5879), 0.25 μM dexamethasone (Sigma, Cat. No. D4902), 10 μg/mL insulin (Sigma, Cat. No. I3536), and P. sibiricus extracts and fractions (50 μg/mL, soln. in DMSO) or pure compounds (10, 20 μg/mL, soln. in DMSO) [63 (link)]. The control group was cultured in the basic maintenance medium without P. sibiricus extracts, fractions or pure compounds. At the end of 6 day, the cells were washed with 0.1 M phosphate buffer solution (pH 7.4), lysed with 1% Triton™ X-100 (BioUltra, Cat. No. 93443), and total lipids were extracted accordingly Bligh-Dyer method [64 (link)]. Triacylglycerol content was determined spectrophotometrically (540 nm) using Sigma kit (Cat. No. TR0100) according to manufacturer’s instruction.

3T3-L1 pre-adipocytes were purchased from the Bioresource Collection and Research Center (BCRC). Dulbecco’s modified Eagle’s medium (DMEM) - high glucose was obtained from Gibco (Waltham, Thermo Fisher Scientific, USA) 11965092. FBS was bought from Gibco (Waltham, Thermo Fisher Scientific, USA). IBMX was bought from Sigma-Aldrich, Inc. (St. Louis, MO, USA) I5879. Dexamethasone was obtained from Sigma-Aldrich, Inc. (St. Louis, MO, USA) D2915, Lot#031M1358V. Insulin was purchased from Sigma-Aldrich, Inc. (St. Louis, MO, USA) DIF001A.
3T3-L1 pre-adipocytes were cultured in DMEM - high glucose supplemented with 10% fetal bovine serum (FBS-DMEM) at 37°C in a 5% CO₂ humidified atmosphere. For adipocyte differentiation, two-day post-confluent cells were placed in 10% FBS-DMEM with 250 nM Dexamethasone, 0.5 mM IBMX, and Insulin (1 μg/ml). After cultured for two days, the cells were cultured in 10% FBS-DMEM containing Insulin (1 μg/ml) for two more days to induce cells toward adipogenesis. The cells were then called 3T3-L1 adipocytes and maintained in 10% FBS-DMEM for later experiments.