3t3 l1 preadipocytes

3T3‐L1 preadipocytes, purchased from RIKEN Bioresource Center (Ibaraki, Japan), were cultured in maintenance medium, Dulbecco's Modified Eagle's Medium (low‐glucose) (Wako, Osaka, Japan) with 10% fetal bovine serum (Thermo Fisher Scientific, Waltham, MA, USA) and 1% penicillin/streptomycin (Sigma‐Aldrich, St. Louis, MO, USA). The differentiation of 3T3‐L1 preadipocytes into mature adipocytes was performed as follows. Preadipocytes were cultured to reach confluence. At confluence, the maintenance medium was changed to adipocyte differentiation medium [the maintenance medium supplemented with 500 μm 3‐isobutyl‐1‐methylxanthine (Sigma‐Aldrich) and 1 μm dexamethasone (Sigma‐Aldrich)], and the cells were then cultured for another 2 days. Subsequently, the adipocyte differentiation medium was changed to adipocyte maturation medium [the maintenance medium supplemented with 10 μg·mL⁻¹ insulin (Sigma‐Aldrich) and 50 nm triiodothyronine (Sigma‐Aldrich)], which was exchanged at 1‐day intervals. For this study, 3T3‐L1 adipocytes were differentiated for 7 days and treated with the indicated reagents for 24 h.

The 3T3-L1 preadipocytes were purchased from Bioresource Collection and Research Center (Hsinchu, Taiwan). The cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) containing 10% fetal bovine serum (FBS), 1% penicillin/streptomycin, and 1 mM sodium pyruvate at 37 °C in 5% CO₂ incubator. Two days after confluence, the cells were cultured in FBS-containing DMEM with the incorporation of 500 μM 3-isobutyl-1-methylxanthine, 0.25 μM dexamethasone, and 10 μg/mL bovine insulin. After 2 days, the medium was changed to DMEM supplemented with 10% FBS and 10 μg/mL insulin. This was continued to day 8 when the preadipocytes had fully differentiated to adipocytes.

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.

The 3T3-L1 preadipocytes (RIKEN Bio Resource Research Center, Tsukuba, Japan) were cultured and maintained in Dulbecco’s modified Eagle’s medium (DMEM; Thermo Fisher Scientific K.K., Tokyo, Japan) containing 3.7 g/L sodium bicarbonate (Fujifilm Wako Pure Chemical, Osaka, Japan), 50 IU/mL penicillin–50 µg/mL streptomycin (Meiji Seika Pharma, Tokyo, Japan), and 10% fetal bovine serum (FBS; Fujifilm Wako) as a normal medium.

3T3-L1 preadipocytes (from Bioresource Collection and Research Center, Food Industry Research and Development Institute, Hsinchu, Taiwan) were maintained in DMEM containing 10% FBS, 3.7 g /L NaHCO₃, 25 mM HEPES, 1 g/L glucose and 100 U/mL penicillin/streptomycin at 37°C, 5% CO₂ and 95% humidity. Two days after confluence, preadipocytes were induced differentiation by incubating cells with DMEM containing 0.5 mM IBMX, 1 μM dexamethasone, 1.7 μM insulin, and 10% FBS at 37°C in a 5% CO₂ humidified incubator for 72 hours. On day 3, the medium was replaced with fresh medium containing 1.7 μM insulin. After day 5, the culture medium was replaced every two days [18 (link), 20 (link)].