B 27 supplement minus antioxidants

OHCs were conducted on 8–10-day-old Sprague-Dawley rats supplied by the animal facilities of Universidad Autónoma de Madrid (Madrid, Spain). Cultures were prepared according to the methods described by Stoppini et al. [52 (link)] with some modifications. Briefly, 300-µm-thick hippocampal slices were prepared from rat pups using a McIlwain tissue chopper and separated in ice-cold Hank’s balanced salt solution (HBSS) composed of (mM): glucose 15, CaCl₂ 1.3, KCl 5.36, NaCl 137.93, KH₂PO₄ 0.44, Na₂HPO₄ 0.34, MgCl₂ 0.49, MgSO₄ 0.44, NaHCO₃ 4.1, and HEPES 25; 100 U/mL penicillin and 0.100 mg/mL gentamicin. Approximately 4–6 slices were placed on Millicell 0.4-µm culture insert (Millipore, Burlington, MA, USA) within each well of a six-well culture tray with the medium, where they remained for 7 days. The culture medium, which consisted of 50% minimal essential medium, 25% HBSS, and 25% heat-inactivated horse serum, was purchased from Life Technologies (Madrid, Spain). The medium was supplemented with 3.7 mg/mL d-glucose, 2 mmol/L l-glutamine, 2% of B-27 Supplement Minus antioxidants (Life Technologies, Madrid, Spain), and 100 U/mL penicillin. OHCs were cultivated in a humidified atmosphere at 37 °C and 5% CO₂, and the medium was changed twice a week.