PC12 Cell Culture and Differentiation

Because of the clonal instability of the PC12 cell line (Fujita et al. 1989 (link)), the experiments were performed on cells that had undergone fewer than five passages, and all studies were repeated several times with different batches of cells. As described previously (Crumpton et al. 2000a (link); Qiao et al. 2003 (link); Song et al. 1998 (link)), PC12 cells (1721-CRL; American Type Culture Collection, Manassas, VA) were seeded onto 100-mm poly-d-lysine-coated plates in RPMI-1640 medium (Invitrogen, Carlsbad, CA) supplemented with 10% inactivated horse serum (Sigma Chemical Co., St. Louis, MO), 5% fetal bovine serum (Sigma Chemical Co.), and 50 μg/mL penicillin streptomycin (Invitrogen). Cells were incubated with 7.5% CO₂ at 37°C, and the medium was changed every 2 days. For studies in the undifferentiated state, cells were seeded at varying densities so that, regardless of the total time of incubation, the cells would reach a final confluence of 60–70%. Twenty-four hours after seeding, the medium was changed to include the various test substances: chlorpyrifos (Chem Service, West Chester, PA), diazinon (Chem Service), parathion (Chem Service), physostigmine (Sigma Chemical Co.), dieldrin (Chem Service), or NiCl₂ (Sigma Chemical Co.). Because of their poor water solubility, the pesticides were dissolved in dimethyl sulfoxide (Sigma Chemical Co.), achieving a final concentration of 0.1% in the culture medium; accordingly, all cultures included this vehicle, which had no effect on the PC12 cells (Qiao et al. 2001 (link), 2003 (link); Song et al. 1998 (link)).
For studies in differentiating cells, 3 × 106 cells were seeded; 24 hr later, the medium was changed to include 50 ng/mL 2.5 S murine NGF (Invitrogen), and each culture was examined under a microscope to verify the subsequent outgrowth of neurites. The test agents were added concurrently with the start of NGF treatment.