PC12 cells were from the American Type Culture Collection (ATCC, Manassas, VA; PC12 cells catalog no. CRL-1721); F12K cell culture medium from Gibco Life Technologies (Grand Islands, NY); tolcapone (to block catechol-O-methyltransferase) from Orion Pharma (Espoo, Finland); DOPAL standard from Santa Cruz Biotechnology, Inc. (Dallas, TX); and Cys-DA standard from the NIMH Chemical Synthesis and Drug Supply Program (No. C-805).
Non-adherent, non-differentiated cells PC12 cells were kept frozen in liquid nitrogen until passaged for experiments. The cells were grown in F12K medium with 15% horse serum and 2.5% fetal bovine serum and incubated at 37 °C in an atmosphere of 5% carbon dioxide. The medium was replaced several times per week and cells passaged once per week.
At 24 hours prior to plating for experiments, the cells were centrifuged and the medium was replaced with medium containing 10 mM tolcapone. After the 24 hours, the cells were collected, suspended in the same medium, and counted (Cellometer, Nexcelom Bioscience, Lawrence, MA). About 500,000 cells/well were plated in 12-well plates. The experiments began after 24 hours of incubation in tolcapone-containing medium.
Non-adherent, non-differentiated cells PC12 cells were kept frozen in liquid nitrogen until passaged for experiments. The cells were grown in F12K medium with 15% horse serum and 2.5% fetal bovine serum and incubated at 37 °C in an atmosphere of 5% carbon dioxide. The medium was replaced several times per week and cells passaged once per week.
At 24 hours prior to plating for experiments, the cells were centrifuged and the medium was replaced with medium containing 10 mM tolcapone. After the 24 hours, the cells were collected, suspended in the same medium, and counted (Cellometer, Nexcelom Bioscience, Lawrence, MA). About 500,000 cells/well were plated in 12-well plates. The experiments began after 24 hours of incubation in tolcapone-containing medium.