3 isobutyl 1 methylxanthine

After achieving 90% cell confluence, the cells were passaged to 12-well plates and cultured until achieving 90% confluence yet again. The basic medium was then removed and replaced with differentiation medium (0.25 µM dexamethasone, 10 µg/ml insulin, and 0.5 mM 3-isobutyl-1-methylxanthine; all from Takara Bio Inc.) for 48 hr. The differentiation medium was replaced with maintenance medium (10 µg/ml insulin; Takara Bio Inc.) and incubated for 48 hr. The detailed procedure for induction of abdominal preadipocytes is outlined in Figure 1. Cells were collected after being induced for 0, 48, 96, and 144 hr (0, 2, 4, and 6 d). Each interval included three biological replicates (n = 3).

Mouse 3T3-L1 preadipocytes were purchased from the American Type Culture Collection (ATCC, Manassas, VA, U.S.A.) and cultured in DMEM supplemented with 10% FBS at 37°C in a humidified atmosphere of 5% CO₂. To differentiate 3T3-L1 into mature adipocytes, the cells were seeded into 6-well plates (Nunc, Roskilde, Denmark) at a concentration of 8 × 10⁴ cells per well, and the medium was replaced after 2 days. At 2 days after confluence, 3T3-L1 cells were transferred to adipogenic differentiation medium, which is DMEM containing 10% FBS and AdipoInducer Reagent (10 μg/ml insulin, 2.5 μM dexamethasone, and 0.5 mM 3-isobutyl-1-methylxanthine; Takara Bio Inc., Shiga, Japan), for 2 days. After that, the cells were cultured in adipocyte maintenance medium, which is DMEM containing 10% FBS and 10 μg/ml insulin, for 2 days. To evaluate the effect of HODE isomers in 3T3-L1 cells, the cells were cultured in the medium containing 12 μg/ml HODEs throughout the experiment.