2.1. Cell Culture

Cerebral cortex neurons in primary culture were used in this study. Cortical cells were chosen because the cortical area is one of the parts of the brain most frequently affected by stroke. Moreover, the neurons in this area contain a large number of glutamate receptors, so it is prone to excitotoxicity, which plays an important role in stroke [3]. Primary cortical cells were obtained from the cerebral hemisphere of embryos of Wistar rats (Harlan, Spain) after 16–18 days of gestation. The rats were killed with CO₂ and the foetuses were decapitated. Dissociated cortical cells were plated in cell culture plates at a density of 10⁵ cells/cm² in 9.6 cm² Corning Costar cell culture plates (Madison, WI, USA). Previous to its use, each well was coated with a 1 mL poly-l-ornithine solution (0.01%) for 1 h in the incubator at 37 °C, the poly-l-ornithine solution was removed and washed three times with sterile water. Once the water was removed, the wells were dried in the sterile chamber with ultraviolet light. The cells were cultured in conditioned Neurobasal Medium^® (Gibco, Waltham, MA, USA) containing penicillin/streptomycin, enriched with GlutaMAX^® (Gibco) and B27^® (containing antioxidants), supplemented with 5% horse serum, 5% foetal bovine serum, and 20 mM glucose. Cultures were incubated at 37 °C in a humidified atmosphere containing 5% CO₂/95% air (pH 7.2). To halt the proliferation of non-neuronal cells, three days after plating, half the culture medium was replaced with medium, as previously described, containing cytosine arabinoside (final concentration: 10 µM). On day 5 after plating, half of the medium was changed with conditioned Neurobasal Medium containing 2 mM glutamine and 2% B27 (AO+), and the neurons were used on day 9.