*2.8. Differentiation of P19 Cells into P19-Derived Neurons*

Exponentially grown cultures were trypsinized and dissociated into single cells. P19 cells (2 × 106 cells/mL) were then suspended in 10 mL <sup>α</sup>-MEM supplemented with 5% FBS, 1% antibiotics-antimycotic solution and 0.5 μM all-*trans*-retinoic acid (RA) and seeded onto a 100-mm bacteriological culture dish. The cells formed large aggregates in suspension. After 4 days of RA treatment, aggregates were dissociated by 5-mL glass measuring pipette, re-plated on poly-L-lysine-pre-coated multi-well plates (multi-well plates were coated with 50 μg/mL poly-L-lysine dissolved in PBS for overnight and sterile under UV light for 30 min) at 7 × <sup>10</sup><sup>4</sup> cells/mL (150 <sup>μ</sup>L/well in 96-well plate), in <sup>α</sup>-MEM supplemented with 10% FBS, and 1% antibiotics-antimycotic solution and incubated for 24 h. Cytosine-1-β-D-arabinoside or Ara-C (10 μM) was added at day 1 after plating and the medium was changed every 2–3 days. The differentiated neuronal cells, P19-derived neurons, were used after day 14 of the differentiation process [18–20].
