Mouse metabolic hormone magnetic bead panel

Blood samples were obtained by cardiac puncture under terminal anesthesia and centrifuged at 3000g for 20 min at 4 °C to collect serum. The serum concentrations of insulin were measured using a Mouse Metabolic Hormone Magnetic Bead Panel (Merck Millipore, Burlington, MA, USA). The serum concentrations of TG, total cholesterol, low-density lipoprotein (LDL)-cholesterol, and high-density lipoprotein (HDL)-cholesterol; and the activities of aspartate aminotransferase (AST) and alanine aminotransferase (ALT) were determined using colorimetric assay kits (Roche).
Cell culture 3T3-L1 preadipocytes were purchased from the American Type Culture Collection (Manassas, VA, USA) and cultured in Dulbecco's modified Eagle's medium (DMEM) containing 10% bovine calf serum (BS) and 1% penicillin/streptomycin (P/S) until confluent. After 2 days (D0), this medium was replaced with DMEM containing 10% fetal bovine serum (FBS), 1% P/S, and MDI (0.5 mM 3-isobutyl-1-methylxanthine, 1 μM dexamethasone, and 4 μg mL -1 insulin). On D2, this medium was replaced with DMEM containing 10% FBS, 1% P/S, and 4 μg mL -1 insulin, and this was replaced every 2 days until day 8 (D8). For OKC treatment, 2-day confluent 3T3-L1 cells were incubated with different doses of OKC (0, 12, 25, 50, or 100 μg mL -1 ) every 2 days during differentiation and until mature adipocyte formation (D8).