Pups were decapitated, and the cerebral cortex was removed and enzymatically dissociated. Cortical neurons were plated on poly‐L‐lysine‐coated (0.1 mg/ml) glass coverslips (Thermo‐Fischer Scientific) at a density of 40,000 cells/mL and maintained up to 14 days in vitro (DIV) at 37°C, 5% CO2, 95% humidity in a culture medium consisting of Neurobasal A, B‐27 (2%), GlutaMAX (1%), and penicillin/streptomycin (1%). All chemicals were purchased from Life Technologies/Thermo‐Fischer Scientific unless stated otherwise.
For the experiments aimed at studying the autophagic flux, 30 nM rapamycin (LC Laboratories, 72 h), 300 nM Bafilomycin A1 (BafA1, 8 h), 100 mM trehalose (72 h), 2 mM 3‐methyladenine (3‐MA, 72 h) (Sigma‐Aldrich, Milano, Italy) treatments were carried. As a control for both treatments, cultures were subjected to the same medium change with addition of an equivalent volume of vehicle.
The production of VSV‐pseudotyped third‐generation lentiviral particles was performed as previously described (Rocchi et al., 2019 (link)). pLenti‐PGK‐Cre‐EGFP or pLenti‐PGK‐ΔCre‐ EGFP plasmids were obtained as previously described (Kaeser et al., 2011 (link)). Primary neurons were infected at 7 DIV at a multiplicity of infection of 10. After 24 h, half of the medium was replaced with fresh medium.
For the experiments aimed at studying the autophagic flux, 30 nM rapamycin (LC Laboratories, 72 h), 300 nM Bafilomycin A1 (BafA1, 8 h), 100 mM trehalose (72 h), 2 mM 3‐methyladenine (3‐MA, 72 h) (Sigma‐Aldrich, Milano, Italy) treatments were carried. As a control for both treatments, cultures were subjected to the same medium change with addition of an equivalent volume of vehicle.
The production of VSV‐pseudotyped third‐generation lentiviral particles was performed as previously described (Rocchi et al., 2019 (link)). pLenti‐PGK‐Cre‐EGFP or pLenti‐PGK‐ΔCre‐ EGFP plasmids were obtained as previously described (Kaeser et al., 2011 (link)). Primary neurons were infected at 7 DIV at a multiplicity of infection of 10. After 24 h, half of the medium was replaced with fresh medium.
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