L glutamine and antibiotics

Immortalized human keratinocytes (HaCaT, Innoprot, Spain) were cultured in Dulbecco’s Modified Eagle’s Medium (EuroClone), supplemented with 10% foetal bovine serum (HyClone), 2 mM l-glutamine and antibiotics (EuroClone) in a 5% CO₂ humidified atmosphere at 37°C. HaCaT cells were cultured following the protocol described by Ferraro et al. (19 (link)).

Animals were treated according to the institutional guidelines, in compliance with the European Community Council Directive 2010/63/UE for care and use of experimental animals. Authorization for animal experimentation was obtained from the local ethical committee on November 10, 2017 and was communicated to the Italian Ministry of Health, in compliance with the Italian law D. Lgs. 116/92 and the L. 96/2013, art. 13. All efforts were made to minimize animal suffering and to reduce the number of animals used. Hippocampal neurons were prepared from postnatal day 0 to 1 (P0–P1) Wistar rats as previously described (Baj et al., 2014 (link)). Cultures were maintained in Neurobasal medium (Life Technologies) supplemented with B27 (Thermo Fisher Scientific, Waltham, MA, United States), 1 mM L-glutamine and antibiotics (Euroclone, Milan, Italy) on 24-well imaging plates (Eppendorf, Hamburg, Germany) or glass coverslips pre-coated with poly-L-ornithine (100 μg/ml) and Matrigel^TM (Corning, NY, United States). At days in vitro 3 (DIV3), cytosine 2.5 μM β-D-arabinofuranoside was added. For transfection experiments, neurons were used at a concentration of 200 cells/mm², used at DIV6 and analyzed 24 h later. For neurite outgrowth analysis, neurons were seeded at a concentration of 320 cell/mm².