Collagen 3D matrices

Neurons were grown in 3D collagen matrices as described previously at a density of 0.75 × 10⁶ to 1.5 × 10⁶ cells/ml (24). For the collagen matrix, 225 μl of collagen (Merck Millipore, catalog no. 08-155) with a concentration of 4.13 mg/ml was combined with 30 μl of 10× MEM and 15 μl of 5.5% sodium bicarbonate and mixed. Transfected cells in medium were added 1:3 and mixed carefully. From the hydrogel-cell mix, single 40-μl drops were added to the bottom of eight-well glass-bottom dishes (ibidi). Gels were kept in a cell culture incubator at 37°C for 20 min before the addition of 400 μl of conditioned N2 medium to fully cover gels.