Cytosine arabinofuranoside

Primary cultures of cerebellar enriched in granule cells were prepared from 8-day-old Sprague-Dawley rats [22 (link), 23 (link)]. Cerebellar granule cells were seeded, respectively, in 96-well microplates (1.0 × 10⁵ cells per well) dishes coated with poly-L-lysine (50 μg/mL; Sigma, St. Louis, MO, USA). The cells were resuspended in the following culture media: DMEM, 10% heat-inactivated fetal bovine serum, glutamine (2.0 mM), gentamicin (100 μg/mL), antibiotic-antimycotic solution (10 μg/mL; Sigma), and potassium chloride (25 mM). After seeding the cells, incubated in a humidified atmosphere of 5% CO₂/95% air at 37°C for 6-7 days. Cytosine arabinofuranoside (final concentration, 10 μM; Sigma) was added after 16 h of culture to inhibit the replication of nonneuronal cells.

Primary cultures of CGNs were prepared from 7-day-old
pups of Wistar rats (Rattus norvegicus). All animal experiments were authorized by the University of Bologna
Bioethical Committee (Protocol no. 1088; Code 2DBFE.N.BFY) and performed
according to Italian and European Community laws on the use of animals
for experimental purposes. For cerebellar granule cultures, cells
were dissociated from cerebella and plated on 35 mm Ø dishes,
previously coated with 10 μg/mL poly-l-lysine, at a
density of 1.2 × 10⁶ cells/2 mL of medium BME supplemented
with 100 mL/L heat-inactivated FBS (Life Technologies), 2 mmol/L glutamine,
100 μmol/L gentamicin sulfate, and 25 mmol/L KCl (all from Sigma-Aldrich);
16 h later, 10 μM cytosine arabinofuranoside (Sigma-Aldrich)
was added to avoid glial proliferation. After 7 days in vitro, differentiated
neurons were shifted to serum-free BME medium containing 25 mmol/L
KCl without the serum and different treatments were performed. For
neurotoxicity experiments, differentiated CGNs were exposed to 10 μM
concentration of the studied compounds for 24 h in serum-free BME
(25 mM KCl). The experiment was performed in triplicate. Since the
native fluorescence of the library could interfere with the reading
of the classical MTT cell viability assay, the Hoechst 33258 staining
was performed to count healthy and apoptotic nuclei.

Human neuroblastoma SH-SY5Y cells were differentiated on different surfaces (Cultrex-coated glass, laminin-coated glass, laminin-coated or not PLLA-FILM, laminin-coated or not random and aligned electrospun PLLA scaffold) using 10 μM RA and cultured for 7 days.
Primary neurons were isolated from newborn mice cortex and cytosine-arabinofuranoside (10 μM; Sigma) was added to the culture medium, to inhibit proliferation of the glial cells,^{7 (link)} and cultured for 15 days.
NSCs were obtained from embryos forebrain and secondary neurospheres were dissociated and plated at a density of 1 × 10⁴ cells/cm² in the same culture medium without mitogens.^{8 (link),9 (link)} To achieve full lineage commitment and cell differentiation, seeded cells were cultured for 15 days.
Animal care and treatment were in accordance with the EU Directive 2010/63/EU and approved by the Ethics Committee of Animal Experimentation, University of Bologna.

Primary neuronal cells were prepared from Long-Evans rat embryos on the 18th day of gestation as described previously [20 (link)]. Brainstems from the fetal brains were dissected out, the tissues were triturated by repeatedly pipetting, and the isolated cells cultured in a serum-free neurobasal medium supplemented with B-27 at a density of 5 × 10⁴ cells/well in 24-well plate. After incubation for 3 days, half of the medium was replaced with fresh medium with cytosine arabinofuranoside (10 μM; Sigma) but without l-glutamic acid, and additional incubation at 37°C was carried out. Six days after seeding, the cultured primary neuronal cells were treated with 10 nM E2, PPT (ERα agonist), and DPN (ERβ agonist) for 24 h, respectively.