Poly l lysine treated

Following a protocol developed by Rescan and colleagues (1995) , primary myoblasts were isolated from juvenile rainbow trout (1-2.5 g). Following mechanical dissociation, white muscle tissue was washed, enzymatically digested (collagenase type IV and trypsin) and cells were filtered (100 and 40 μm). Isolated cells were counted using a hemocytometer and the trypan blue exclusion method. Isolated cells were plated on poly-L-lysine-treated (Sigma), laminin-coated (BD Biosciences) plates at a density of 2x10⁶ cells/mL. Cultures were incubated at 18 °C in complete media (10% DMEM) under normal atmospheric conditions without CO₂ supplementation. Media was changed daily for the first two days of culture. On day three, cells were treated with media containing cortisol or ethanol (vehicle control). All treatments were run in triplicate on duplicate plates and consisted of increasing concentrations of cortisol: CORT 0, 10, 100, and 1000 ng/mL. After 24 hr, media was removed and cells were harvested for total RNA isolation (RNAzol; Molecular Research Center).

Primary neurons were isolated from E18 Sprague–Dawley rat embryos (Harlan Laboratories, Indianapolis, IN, USA). Rat pups were euthanized and were placed in ice-cold isotonic solution [composition (g/L): NaCl, 8; KCl, 0.4; KH₂PO₄, 0.03; glucose, 0.1; and sucrose, 20.2; in dH₂O at pH 7.4; all components from Sigma-Aldrich, St. Louis, MO, USA]. After removing meninges and blood vessels, brains were placed in an isotonic solution of 0.25% trypsin–EDTA (Cellgro #25-053-CI; Mediatech, Manassas, VA, USA) at 37ºC for 10 min to disrupt the connective tissue. Trypsinization was then stopped using DMEM complete [DMEM+10% fetal calf serum + antibiotic–antimycotic (Invitrogen, Carlsbad, CA, USA)], and triturated through a fire-polished Pasteur pipette (Fisher Scientific, Waltham, MA, USA) until the solution became much more uniform. Supernatant was aspirated into another tube and centrifuged at 126 × g (1,500 rpm) for 10 min and then resuspended in DMEM complete. The cells were plated at 2.5 × 10⁵ cell/cm² onto poly-L-lysine-treated (Sigma-Aldrich) 24-well plates (Corning^® Life Sciences, Corning, NY, USA). Media was changed to Neurobasal (#21003-049; Invitrogen, Carlsbad, CA, USA) supplemented with B-27 (#17504-044; Invitrogen) the following day and every second day thereafter for 7 days.