2.3. PC12 cell cultures and treatments

Because of the clonal instability of the PC12 cell line (Fujita et al. 1989), the experiments were performed on cells that had undergone fewer than five passages. As described previously (Qiao et al. 2003; Song et al. 1998), PC12 cells (American Type Culture Collection CRL-1721, obtained from the Duke Comprehensive Cancer Center, Durham, NC) were seeded onto poly-D-lysine-coated plates in RPMI-1640 medium (Sigma) supplemented with 10% horse serum (Sigma), 5% fetal bovine serum (Sigma), and 50 μg/ml penicillin streptomycin (Invitrogen, Carlsbad, CA). Incubations were carried out with 5% CO₂ at 37°C, standard conditions for PC12 cells. Twenty-four hours after plating, we initiated neurodifferentiation (Jameson et al. 2006b; Slotkin et al. 2007; Teng and Greene 1994) by changing the medium to include 50 ng/ml of 2.5 S murine nerve growth factor (Promega Corporation, Madison, WI). Test substances, dissolved in DMSO, were added simultaneously so as to be present throughout neurodifferentiation. Control samples contained the corresponding final concentration of DMSO vehicle (0.1%), which has no effect on PC12 cell viability or differentiation (Qiao et al. 2001, 2003; Song et al. 1998). The medium was changed every 48 hr with the continued inclusion of nerve growth factor and test substances, and continued for 6 days, parallel to the exposure used for NSCs. At the end of the 6 day exposure period, the cultures were examined under a microscope to verify the outgrowth of neurites.