4.2.2. CBA Screening Assay

For cell maintenance, neuro-2a (N2a) cells (ATCC, CCL131) were cultured in 10% fetal bovine serum (FBS) RPMI medium with 1% sodium pyruvate solution (100 mM), 1% l-glutamine solution (200 mM), and 0.5% antibiotic solution (10 mg/mL streptomycin and 1000 U/mL penicillin) (Sigma-Aldrich, St. Louis, MO, USA). Cultures were maintained at 37 °C and 5% CO₂ in a humid atmosphere incubator (Binder, Tuttlingen, Germany). The day prior to the assay, cells were seeded in a 96-well microplate in 200 μL of 5% FBS-RPMI medium at a density of 35,000 cells per well. Cells were incubated under the same conditions as described for cell maintenance. Every standard (CTX-1B) and sample extract were assayed in triplicate. Half the wells of each microplate were pretreated with ouabain and veratridine corresponding to a final concentration of 1 and 0.1 mM, respectively. Standard solutions and sample extracts were reconstituted in RPMI 5%. Then, samples were serially diluted, and 10 μL added to each well, with (O/V+) and without (O/V-) ouabain and veratridine pretreatment. According to matrix effects estimated without (O/V-), concentrations for the different fish extracts were adjusted to avoid matrix effects. On the next day, cell viability was measured, by means of the MTT test [3-(4,5-dimethylthiadol-2-yl)-2,5-diphenyltetrazolium] (500 μg/mL) [35] and absorbance measured at 570 nm using an automated multi-well scanning. Samples were considered to be positive when cell viability was inhibited in O/V+ wells and unaffected in O/V- wells.