Rat pheochromocytoma pc12 cells

Rat pheochromocytoma (PC12) cells were purchased from American Type Culture Collection (Manassas, VA, USA) and were maintained in DMEM F12 medium (Invitrogen, Grand Island, NY, USA) supplemented with 5% FBS (Thermo Scientific, Waltham, MA, USA), 10% horse serum, L-glutamine (2 mM), penicillin (100 U/ml) and streptomycin (100 µg/ml) in a humidified atmosphere (5% CO 2 ) at 37°C. Cells were subcultured three times a week at a density of 500000/ml and the differentiation was achieved by treatment with 50 ng/ml nerve growth factor (NGF, Sigma, St Louis, MO, USA) for one week (Barbault et al., 2009) . Cells were cultured in DMEM medium (Invitrogen, USA) supplemented with 10% FBS (Thermo Scientific, USA) and the cultures were maintained at 37°C in a humidified atmosphere with 5% CO 2 (Reale et al., 2014) . The N9 murine microglial cell lines were cultured in Dulbecco's modified Eagle medium (DMEM) supplemented with 10% heat inactivated FBS, penicillin (100 U/ml) and streptomycin (100 µg/ml) (Corradin et al., 1993) . Cells were grown in a humidified environment containing 5% CO 2 at a constant temperature of 37°C.
Before the experiments, the cell culture medium was replaced with fresh serum-free medium for another 24 h to minimize the interference of growth factors in the serum with signal transduction.