Cytosine β d arabinofuranoside hydrochloride arac

Cell suspensions were prepared from rat hippocampus as described previously [21 (link)]. Coverslips (for immunofluorescence staining and calcium imaging) and culture dishes (for any other application) were coated with poly-D-lysine hydrobromide (Sigma-Aldrich, Taufkirchen, Germany). Cell suspension was preplated onto an uncoated flask and incubated at 37 °C in 5% CO₂ for 1 h. During this time glial cells settled down and adhered to the bottom of the flask, while neurons remained in the supernatant. Supernatant was collected thereafter and centrifuged at 800 rpm for 8 min at room temperature. Cell pellets were resuspended and cultured in serum-free Neurobasal-A medium supplemented with 2% B27, 0.5 mM GlutaMAX and 1% penicillin-streptomycin (all Life Technologies, Darmstadt, Germany). Cells were plated on poly-D-lysine coated dishes or coverslips at a density of 2.5 × 10⁵ onto 3.5 cm². Cells were maintained at 37 °C in a fully humidified incubator containing 5% CO₂. After 24 h Cytosine β-D-arabinofuranoside hydrochloride (AraC; Sigma-Aldrich) was added to inhibit proliferation of remaining glial cells. Neurons were maintained in dispersed culture with the original media up to 40 days in vitro (DIV).

E15.5–16.5 mouse embryo brains were dissected and washed in ice-cold 0.1 M phosphate-buffered saline (PBS) containing 6.5 mg/mL glucose. The meninges were removed and the cortical lobes isolated. Tissue pieces were trypsinized for 15 min at 37 °C. After addition of horse serum and centrifugation, cells were dissociated by trituration in 0.1 M PBS containing 0.025% DNase with a polished glass pipette (all from Sigma-Aldrich, Darmstadt, Germany). Dissociated cells were plated at ~3000 cells/mm² on plates (Nunc, Denmark) coated with poly-D-lysine (Sigma-Aldrich). The culture medium was Neurobasal^TM supplemented with 2 mM glutamine, 6.5 mg/mL glucose, antibiotics (Pen/Strept), 5% horse serum, and B27 (Invitrogen-Thermo Fisher Scientific, Waltham, MA, USA). After 72 h, 5 µM cytosine β-D-arabinofuranosidehydrochloride (AraC) (Sigma-Aldrich) was added for 48 h to inhibit the growth of dividing non-neuronal cells. Cultures were used after at least 7 days in vitro.
For PrP^C overexpression on cortical cultures, PrP^C-encoding plasmid (pcDNA 3.1 backbone), provided by D. A. Harris (Boston University School of Medicine, Boston, MA, USA) was transfected using Lipofectamine 2000 (Invitrogen-Thermo Fisher Scientific), according to the manufacturer’s instructions.

Hanks' balanced salt solution (HBSS), serum-free neurobasal media (NB), B27 supplement (50x), L-glutamine, Prolong Gold anti-fade reagent with 4′,6-diamidino-2-phenylindole (DAPI), SuperScript III reverse transcriptase, SYBR/Green Master mix, and Alexa 405-, 488-, or 555-conjugated secondary antibodies were obtained from ThermoFisher. Poly-L-lysine hydrobromide, cytosine β-D-arabinofuranoside hydrochloride (Ara-C), and anti-actin antibody were from Sigma. Microtubule-associated protein 2 (MAP-2) antibody was obtained from Abcam. C5aR1 antibody (10/92) was obtained from SeroTec (Ager et al., 2010 (link)). Anti VGLUT1 and anti GAD67 antibodies were obtained from Millipore. HRP-conjugated F(ab')₂ antibodies were from Jackson ImmunoResearch Laboratories. Human C5a peptide was obtained from CompTech. PMX53 was obtained from Cephalon. All buffers are made with Millipore-purified water with additional filters to eliminate LPS and are periodically tested for endotoxin using the Limulus amebocyte lysate clot assay (all solutions added to cells were <0.1 EU/mL; 1 EU is equivalent to 0.1 ng/mL LPS).