Papain dissociation solution

Mouse primary hippocampal neuronal cultures were prepared as previously described (Encalada et al., 2011 (link)). Briefly, hippocampi were dissected from P2 mouse pups in cold Hanks’ balanced salt solution (HBSS) supplemented with 0.08% D-glucose (Sigma-Aldrich), 0.17% Hepes, and 1% penicillin-streptomycin (Pen-Strep); filter-sterilized; and adjusted to pH 7.3. After dissection, the hippocampi were washed twice with cold HBSS and individually incubated at 37°C for 20 min in papain dissociation solution 45 U of papain (Worthington), 0.01% deoxyribonuclease (DNase), 1 mg of DL-cysteine, 1 mg of bovine serum albumin (BSA), and 25 mg of D-glucose (all from Sigma-Aldrich) in phosphate-buffered saline (PBS). After digestion, the hippocampi were washed twice with DMEM, preheated to 37°C, supplemented with 10% FBS, and dissociated by 10 cycles of aspiration through a micropipette tip. Dissociated neurons were then resuspended in warm DMEM supplemented with 10% FBS and plated in six-well plates containing 25-mm sonicated glass coverslips pretreated with 50 μg/ml poly-L-lysine (PLL; Sigma-Aldrich). After 1 h, the medium was replaced with Neurobasal-A medium, which was supplemented with 2% B-27 and 0.25% GlutaMAX (neuronal medium). All reagents were from Gibco except when otherwise indicated. Primary neurons were maintained in a standard tissue culture incubator at 37°C with 5.5% CO₂.