Primary neuronal culture.

All of the reagents for primary neuron culture were from Thermo Fisher Scientific unless indicated otherwise. Primary cortico-hippocampal cultures for prion toxicity studies were prepared from embryonic day 17 FVB mouse brains. Cortices and hippocampi were dissected in chilled dissection medium consisting of calcium- and magnesium-free Hanks’s Balanced Salt Solution, 10 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid, 2 mM L-glutamine and 100 U/mL of penicillin–streptomycin. Brain tissue was next treated with 0.25% (wt/vol) trypsin and 1,000 U of benzonase (VWR) for 15 min at 37 °C and washed three times with dissection medium. Cells were then resuspended in the plating medium consisting of Dulbecco’s Modified Eagle’s Medium supplemented with 10% (vol/vol) of heat-inactivated horse serum and 20 U/mL of penicillin–streptomycin. Next, the cells were triturated with fire-polished glass Pasteur pipettes with two different sizes of opening, filtered through 70-µm strainers, counted using a disposable Neubauer hemocytometer (Labtech), and plated out at 1.5 × 10⁴ cells/well in poly-l-lysine-coated, black µ-clear F-bottomed 96-well plates (Greiner) prefilled with warm plating medium.

Primary cells were left to settle for 1.5 h in a tissue culture incubator (37 °C, 5% CO₂), and afterward the plating medium was changed to warm Neurobasal medium supplemented with 2% (vol/vol) B27, 0.25% (vol/vol) GlutaMAX, and 100 U/mL of penicillin–streptomycin. Primary cells were typically used in the toxicity assays at the age of 10 d in vitro.