Src-deficient Cortical Neuron Cultures

Prenatal pups (embryonic day 17-18) produced from a Src^thl/+ intercross were used to prepare cortical cultures according to previously published protocols (Broeke et al, 2010 (link)) with modifications. All experiments were carried out with Src^thl/thl and WT littermates. Specifically, dissected cortex was digested with 0.025% Trypsin/EDTA at 37 °C in HBSS (Sigma-Aldrich) for 20 min and washed with prewarmed medium to stop digestion. Cells were triturated approximately 10 times with a 1000 μl tip. Neurons were centrifuged at 1000 rpm for 5 min, supernatant was removed and cells resuspended in culture media. Cell density was determined using an Improved Neubauer hemocytometer and low-density cultures were plated on Poly-D-Lysine (0.1 mg/ml; Sigma-Aldrich) coated glass coverslips (18 mm; #1.5, Warner Instruments) at 100,000 neurons/cm². Neurons were maintained in Neurobasal medium (Invitrogen) with 2% B27 (Invitrogen), 1× Penn/Strep (Invitrogen), and 2 mM GlutaMAX (Invitrogen) at 37 °C with 5% CO₂ for 6 days in vitro (DIV6).