Adipocyte isolation and culture

Fifteen to forty grams of AT biopsies were microdissected, rinsed several times in 0.9% NaCl, and digested with 5 ml of Krebs-Ringer solution (0.12 M NaCl, 4.7 M KCl, 2.5 mM CaCl₂, 1.2 mM MgSO₄, 1.2 mM KH₂PO₄) containing 20 mM HEPES pH 7.4, 3.5% fatty acid-free BSA, 200 nM adenosine, 2 mM glucose and collagenase (type 1) for 1 hour (1 mg/g AT) at 37°C in shaking water bath [30]. After collagenase digestion the adipocytes were isolated and cultured in low-glucose Dulbecco's modified Eagle's medium (1,000 mg/L D-(+) -glucose) and ACM was collected after 18 hours. In some experiments, adipocytes were stimulated with DHA or AA. DHA and AA were dissolved under nitrogen condition in 100% ethanol to make 10 mM stock solutions, which were stored at −20°C. Stock solutions were diluted in culture media prior to the cell treatment. Final concentration of ethanol in treated cells was less than 0.1%. To define the lowest effective concentration of DHA (Sigma Aldrich, St. Louis, MO, USA) and AA (Cayman Chemical Company, Ann Arbor, MI USA) able to modulate adipocyte activities, we carried out preliminary experiments, incubating the isolated adipocytes with different concentration of DHA (5–50 μM) and AA (1–25 μM) for different periods of time (6–24 hours). On the basis of the data obtained (not shown), the experiments were carried out incubating the adipocytes with 10 μM DHA and 5 μM AA for 18 hours, respectively.