3t3 l1 adipocytes

For in vitro experiment, mouse 3T3‐L1 adipocytes were ordered from American Type Culture Collection. 3T3‐L1 adipocytes (5 × 10⁴) were cultured and treated with different concentrations of E2. All the experiments were repeated three times.

Schisandrin B (Sch B) was purchased from Ningli Technology Co. Ltd (Kunming, China). 3T3-L1 adipocytes were purchased from American Type Culture Collection (ATCC). Dulbecco’s modified essential medium (DMEM), fetal calf serum, penicillin, streptomycin, Hank’s Balanced Salt Solution (HBSS) and Fluo-3/AM were purchased from Life Technologies Limited. Antibodies for adipocyte triglyceride lipase (ATGL), hormone sensitive lipase (HSL), p-HSL (at serine 563 and serine 565), cleaved caspase 3 and GAPDH were purchased from Cell Signaling or Santa Cruz Biotechnology Inc. Dexamethasone, methyisobutylxanthane, insulin, isoproterenol, free glycerol reagent, glucose, fatty acid free BSA, Oil Red O, Nile Red, hematoxylin and eosin, dimethyl sulfoxide and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) were purchased from Sigma-Aldrich Chemical Co. H89 was purchased Calbiochem. CAY10499 was purchased from Cayman Chemical. All organic solvents were HPLC grade from Sigma-Aldrich Chemical Co.

The C2D cell line was created by our group and was cultured in DMEM₄ as described previously [21] (link), [23] (link), [24] (link).
3T3-L1 adipocytes were obtained from the American Type Culture Collection (Manassas, VA). Adipocytes were cultured and differentiated as described previously [25] (link), [26] (link).
Direct co-culture of 3T3L1 adipocytes and C2D-IL10 or C2D-vector cells were performed by directly adding C2D-IL10 or C2D-vector (0.5×10⁵ viable cells; trypan blue exclusion) to 12-well plates in DMEM₁₀ containing 4 X 10⁵ 3T3L1 cells that had been differentiated for 8 days. Macrophages were incubated with 3T3L1 cells for four days and did not appear apoptotic or necrotic after the 4-day incubation period, as assessed by light microscopic examination.