N methyl d aspartate

Cortical neuronal cultures were prepared as previously described [15 (link)]. E17 rat cortices were isolated, trypsinized, and dissociated in Ca²⁺ and Mg²⁺ free HBSS (Gibco, Carlsbad, CA). Neurons were plated at a density of 1.3 × 10⁵ cells/cm² in polyethyleneimine-coated 24-well plates in Neurobasal medium supplemented with B-27 (Gibco) and 1% antibiotic/antimycotic solution (104 U of penicillin G/ml, 10 mg of streptomycin/ml, and 25 µg of amphotericin B/ml) (Sigma). Cytosine β –D-arabinofuranoside (2.5 µM; Sigma) was added on DIV 3 to prevent the proliferation of astrocytes.
For neuronal death-inducing stimuli, neuronal cultures were incubated on DIV 11 with 10 μM N-methyl-D-aspartate (NMDA) (Tocris) + 1 μM glycine (Sigma), 10 µM 4-hydroxynonenal (Sigma), or 10 nM staurosporine (Sigma) for 24 h. OGD was induced by replacing the conditioned media for glucose and pyruvate-free medium and incubating the cells in a hypoxia chamber (StemCell Technologies, Cambridge, MA) with a 100% N₂ atmosphere for 1 h, at the end of the OGD period, cells were switched to normal Neurobasal + B27 and placed in a ~21% O₂ atmosphere. In the indicated experiments, NPC-EVs with a total amount of 800 ng of protein/mL were added to neuronal cultures for 24 h in combination with the death indicing stimuli, or after OGD.

Drugs used in the experiments include nicotine from Sigma Aldrich (Saint Louis, MO, USA), kainate from Sigma Aldrich (Saint Louis, MO, USA), N-methyl-D-aspartate (NMDA) from Tocris Bioscience (Bristol, UK), 5-hydroxytryptamine (5HT) from Sigma Aldrich (Saint Louis, MO, USA) and irinotecan from Sibudan (Buenos Aires, Argentina).