Co-culturing of neurons with an astrocytic feeder layer

For the co-culture, an astrocyte feeder layer was prepared 21 days in advance from P0/P1 WT mouse pups. Briefly, after isolating the brain and cleaning meninges and choroid plexus, the cortex was removed and put in GGM (Glial Growth Medium: DMEM (Gibco) supplemented with 1.35% glucose, 1% pen/strep and 10% FCS). After addition of pre-warmed 0.25% trypsin (Sigma), the tissue was digested for 15 min at 37°C in a water bath while shaking (300 rpm). After that, 50 µg/mL of DNAse I (Sigma) was added for 1 min and the enzymatic reaction was stopped by adding 4 volumes of GMM. Cells were then washed twice with GGM by centrifuging 5 min at 180xg, the cell suspension was passed through a 70 µm cell strainer and the final cell suspension was plated in T75 flasks with GMM.

For neuronal preparation, P0 mice pups were used as previously described [48]. Briefly, after extracting the brain and cleaning from meninges and choroid plexus, the isolated hippocampi were digested at 37°C for 15 min in dissection media (1× HBSS (Gibco), 1% pen/strep, 10 mM HEPES (Gibco) and 0.6% glucose) containing pre-warmed 0.25% trypsin (Gibco). After the enzymatic digestion, DNAse I (20 µg/mL) was added and incubated at RT for 1 min. GMM was added to quench the enzymatic reaction. Cells were then centrifuged for 5 min at 180xg. The resulting pellet was gently triturated in Neuronal Maintenance Medium (NMM, Neurobasal medium (Gibco) containing 1% glutamax (Gibco), 2% B27 (Gibco) and 1% pen/strep) using a 5 mL pipette followed by a 1 mL pipette. Cells were then centrifuged again for 5 min at 180xg. The pellet was resuspended in NMM and passed through a 70 µm cell strainer. About 40,000 cells were plated in 24-well plates containing pre-coated poly-L-lysin coverslips and NMM. After 2 h, the coverslips were inverted on the top of the pre-cultured feeder layer with a wax-dot spacer in between. After 24 h, the mitotic inhibitor FUDR (2′-Deoxy-5-fluorouridine;Sigma) was added in a concentration of 10 µM. Neuronal cultures were kept at 37°C in 5% CO₂ for 14 days and half of the medium was replaced every 3 days.