Differentiation of PC-12 Cells into Neurons

Rat pheochromocytoma (PC-12) cells were obtained from American Type Culture Collection (ATCC, Manassas, VA, USA). The cells were grown in suspension in RPMI-1640 (ATCC) with 10% heat-inactivated horse serum (Gibco, Grand Island, NY, USA) and 5% fetal bovine serum (Gibco) at 37 °C and 5% CO₂. To differentiate the cells into neuronal cells [26 (link)], 5 × 10⁴ cells were grown for 10 days on collagen-coated dishes (Corning, Corning, NY, USA) in RPMI-1640 with 1% heat-inactivated horse serum and nerve growth factor (NGF) (100 ng/mL, Alomone Labs, Jerusalem, Israel). The medium was changed every two days to fresh differentiation medium. NGF was removed overnight before the day of the experiment. Neuronal morphology and neurite outgrowth were assessed via microscopy before proceeding.