A number of 40 adult specimens of P. lineatus (500–800 g) were obtained from a commercial fish farm (Paulo Lopes City, Santa Catarina State, Brazil; http://www.pisciculturapanama.com.br) and

maintained collectively in 3.000 l water tank with dechlorinated tap water for a period of 3–4 weeks before hepatocytes isolation. Constant aeration was performed by submerged pumps and food was supplied through commercial pelleted fish food (Supra Acqua Line®, 28% of protein) twice a week. Fishes were anesthetized with benzocaine (200 ppm in water), injected PCI-32765 order with 0.5 ml of heparin (5000 U l−1) through the caudal vein and maintained during 5 min in dechlorinated water; then, fishes were anesthetized again and killed by spinal cord section for liver removal. The liver was kept in phosphate buffered saline (PBS, pH 7.6, 4 °C) supplemented with amphotericin-B (25 μg l−1), streptomycin (100 μg ml−1) and penicillin

(100 U ml−1) during 10 min for antibiotic shock and perfused through the portal vein and arterial system with ice-cold PBS-EDTA solution (2 mM EDTA, 1.0 g l−1d-glucose in phosphate buffered saline – PBS, pH 7.6) for blood removal. After perfusion, the liver was aseptically minced with stainless steel blades in PBS containing dispase (1.0 U l−1) and 1.0 g l−1d-glucose, and incubated for 3 h (30 °C) for the hepatocytes dissociation. The cell suspension was forced through a stainless-steel mesh (60–60 mesh) for additional mechanical BMS-354825 mw disruption. Cells were collected, centrifuged at low speed (100–120 g,

3–5 min), washed four times with PBS for debris removal and suspended to a density of 1.0 × 106 cells per ml in RPMI 1640 medium (2.0 g l−1d-glucose, pH 7.6) supplemented buy Sirolimus with NaHCO3 (25 mM), human insulin (0.1 U ml−1), gentamycin (40 mg l−1), streptomycin (10 μg ml-1), penicillin (10 U ml−1), amphotericin-B (2.5 μg l−1) and fetal bovine serum (5% v.v−1). Finally, 2.0 × 105 and 1.0 × 106 cells (viability ⩾97%) were, respectively, seeded onto 96- and 24-well microplates (TTP® or Biofil®) and kept at 24 °C in a CO2 incubator (1.7% of pCO2). For each cell culture, a pool of cells from three fishes was utilized. Before establishing this protocol, non-enzymatic dissociation and several enzymatic digestions were tested: EDTA (2 mM in PBS), trypsin–EDTA (0.05% tripsin, 2 mM EDTA in PBS), pancreatin (0.25% in PBS, 30 min, room temperature), collagenase IV (0.25 U ml−1 in PBS, 30 min, 30 °C), collagenase IV (0.15 U ml−1 in PBS) associated with dispase (0.5 U ml−1 in PBS, 30 min, 30 °C), and dispase (1 U l−1 in PBS, 30 min, 30 °C).