Cytosine d arabinofuranoside

Primary cortical neuronal cultures were established from E16-PND0 mice. Cortices were dissected in Hibernate E (Life Technologies) and digested with 0.25 % trypsin in HBSS (Life Technologies) for 20 min at 37 °C. Tissue was washed three times with HBSS, dissociated in DMEM (Life Technologies) supplemented with 5 % fetal bovine serum (FBS, Genesee), and gently triturated through a glass pipette with a fire-polished tip. Dissociated cells were filtered through a 70 µm cell strainer to remove any residual non-dissociated tissue and plated onto poly-D-lysine-coated 1.5 mm coverglasses or dishes (MatTek) at a density of 4 × 10⁴ cells/cm² for transfection and imaging experiments. For all cultures, media was replaced 3 hours after plating with serum-free Neurobasal-A medium containing B27 supplement (Life Technologies), 2 mM Glutamax (Life Technologies), and 1 % penicillin/streptomycin (Life Technologies) (neuronal growth media). 5 μM cytosine-D-arabinofuranoside (Sigma) was added to the culture medium to inhibit the growth of glial cells three days after plating. Neurons were maintained at 37 °C with 5 % CO₂ and fed twice a week with freshly made culture medium until use.

Primary cultures of dissociated cortical and hippocampal cells were prepared and maintained as previously described [59 (link)]. Briefly, the neocortex and hippocampus of postnatal day 0–1 rat pups were dissociated and plated onto tissue culture plates precoated with poly-L-lysine (molecular weight 300,000, Sigma, St. Louis, MO). For ICC studies, cell suspensions were plated onto CoStar® 96-well plates (Corning, Inc, Corning, NY); for ELISA studies, on Nunc® 6-well polystyrene plates (Thermo Scientific, Waltham, MA). For the ICC and ELISA studies, cortical cells were plated at a density of 78,000 cells/cm² while hippocampal neurons were plated at a density of 31,250 cells/cm². For analysis of network activity, dissociated cells were plated on 12-well polystyrene MEA plates in which each well contained 64 nanoporous platinum electrodes (Axion Biosystems, Inc., Atlanta, GA). MEAs were precoated with poly-L-lysine (0.5 mg/ml, Sigma) and laminin (10 μg/ml, Invitrogen, Carlsbad, CA) and cells were plated at a density of 235,785 cells/cm². All cultures were maintained in Neurobasal-A (Invitrogen) supplemented with 2% B27 (Invitrogen) and 2 mM Glutamax (Invitrogen). At DIV 4, cytosine-D-arabinofuranoside (Sigma) was added to the medium at a final concentration of 5 μM. Half of the medium was replaced with fresh Neurobasal-A supplemented with B27 once weekly.

Hippocampi were dissected from embryonic day 18 rat embryos and dissociated enzymatically for 20 min at 37°C in 0.25% (w/v) trypsin (ThermoFisher Cat# 15050065) in HBSS and dissociated mechanically by triturating with glass polished Pasteur pipettes. Dissociated cells were suspended in plating medium containing Neurobasal (ThermoFisher Cat# 21103049) supplemented with 10% FBS (ThermoFisher Cat# 16140071), 2% B27 (ThermoFisher Cat# 17504044), 2% GlutaMAX (ThermoFisher Cat# 35050061), and 0.001% gentamycin (ThermoFisher Cat# 15710064) and plated at 60,000 cells per dish in glass bottom dishes (MatTek Cat# P35G-1.5–14 C) coated with 0.5 mg/mL poly-L-lysine (Sigma-Aldrich Cat# P2636). At 7 days in vitro (DIV), cytosine-D-arabinofuranoside (Sigma-Aldrich Cat# 251010) was added to a final concentration of 5 µM to inhibit non-neuronal cell growth. Neurons were transiently transfected with 1 µg of each nAb mammalian expression plasmid at 7–10 DIV using Lipofectamine 2000 (ThermoFisher Cat# 11668019) for 1.5 hr as described by the manufacturer. Neurons were used 40–48 hr post transfection for IF-ICC.

EDA was purchased from Aladdin Reagent Co. (Shanghai, China). MTT, fluorescein diacetate (FDA), propidium iodide (PI), 2′,7′-dichlorofluorescein diacetate (DCFH-DA), DNase, poly-L-lysine, and cytosine-D-arabinofuranoside were obtained from Sigma-Aldrich (St. Louis, MO, USA). Basal medium Eagle (BME), fetal bovine serum, penicillin, and streptomycin were purchased from Invitrogen (Carlsbad, CA, USA). Mitochondrial membrane potential assay kit and caspase 3 activity assay kit were obtained from Beyotime (Shanghai, China). The cytotoxicity detection kit (LDH) was purchased from Roche Diagnostics (Mannheim, Germany).