5 6 eet

Primary hepatocytes were isolated by collagenase/EGTA perfusion as published elsewhere (18 ) from male C3HeB/FeJ mice. Following preparation, hepatocytes were resuspended in Williams E Medium (Gibco, Paisley, UK, Catalogue Number 32551), 10% (v/v) FCS, 100 nm dexamethasone. The hepatocyte cell lines Hepa 1–6 and BNL CL.2 were obtained from ATCC (Manassas, VA) and cultured in DMEM (Gibco, Catalogue Number 41966), 10% (v/v) FCS.
For stimulation experiments, cells were seeded onto collagen I coated six-well plates and cultured in serum-free starvation medium (Williams E Medium and DMEM for primary hepatocytes and hepatocyte cell lines, respectively). Subsequently, cells were pre-treated with EETs (4 μm of 5(6)-EET, 8(9)-EET, 11(12)-EET and 14(15)-EET (Cayman Chemicals, Ann Arbor, MI) or vehicle (DMSO) in starvation medium for 1h at 37 °C 5% CO₂. Next, cells were stimulated with starvation medium containing the indicated concentrations of human insulin (Sigma-Aldrich, Steinheim, Germany) with or without EETs at the same concentration as above. Stimulation media for this second step were supplemented with Phosphatase Inhibitor Mixture 2 and 3, (Sigma-Aldrich) at 1:200. DMSO was added to wells without EETs to yield the same final concentration in all wells of the experiment. Cells were incubated for 20, 40 or 60 min at 37 °C 5% CO₂ and scraped into 50 μl RIPA buffer.