Nahco3 7.5

For preparation of primary hippocampal neurons, we used postnatal P0-P2 mice. Briefly, pups were killed by decapitation, and after removing skull and meninges, the hippocampus was dissected and collected in 10mM glucose in PBS containing 0.5 mg/ml papain (Sigma-Aldrich) and 10μg/ml DNAse (Roche). Hippocampi of PrP^CGPIThy-1 mice were collected individually, and the tails were used for genotyping. After 30 min incubation at 37°C, samples were washed 4 times with plating medium (MEM 1X (Gibco), 20mM glucose (Sigma), 10% Horse serum (PAA Laboratories) and 3% of NaHCO₃ 7.5% (Gibco)) and carefully pipetted up and down several times in order to mince the tissue. Cells were plated in 6-well plates containing coverslips previously treated with 0.5 mg/ml of Poly-L-Lys and incubated at 37°C in a 5% CO₂ cell culture incubator. After 4 hours, the media was changed to Neurobasal A medium (Gibco) containing 2% of B27 serum, Glutamax (Gibco) and penicillin/streptomycin (PAA Laboratories). Next day, AraC (Sigma-Aldrich) was added to kill proliferating cells. Half of the media was changed every three days.

P0–P2 mice were used. Briefly, hippocampi were dissected and collected in 10 mM glucose in PBS containing 0.5 mg/ml papain (Sigma-Aldrich) and 10 μg/ml DNAse (Roche Diagnostics). After 30 min incubation at 37 °C, samples were washed with plating medium (MEM 1X (Gibco), 20 mM glucose (Sigma), 10% Horse serum (PAA Laboratories) and 3% NaHCO₃ 7.5% (Gibco)) and plated in 6-well plates containing Poly-L-Lys-pretreated coverslips and incubated at 37 °C in a 5% CO₂ cell culture incubator. After 4 hours, the media was changed to Neurobasal A medium (Gibco) containing 2% of B27 serum, Glutamax (Gibco) and penicillin/streptomycin (PAA Laboratories). Next day, AraC (Sigma-Aldrich) was added in order to kill any proliferating cells. Half of the media was changed every 3 days. Analysis of proteolytic PrP^C shedding in primary neurons was performed as described earlier24 (link).