查看文章

MATERIALS AND METHODS

Materials NCB-20 neuroblastoma cells were kindly provided by Dr. Lovinger (National Institute on Alcohol Abuse and Alcoholism, USA). Proanthocyanidin was purchased from InterHealth Nutraceuticals Inc.(Benicia, CA, USA), as a grape seed proanthocyanidin extract (GSPE) containing approximately 76% oligomeric proanthocyanidins and 3% monomeric bioflavonoids. Cell culture reagents were obtained from Gibco BRL (Rockville, MD, USA). Serotonin and all other chemicals were purchased from Sigma Chemicals (St. Louis, MO, USA).

Cell Culture NCB-20 cells were maintained under conditions previously described. 17) Frozen cell stocks were maintained in liquid nitrogen, and thawed as needed. Cells were routinely grown in medium containing 90% Dulbecco’s modified Eagle’s medium, which contained 3.7 g/l NaHCO3 (pH adjusted to 7.4 with NaOH), 10% fetal bovine serum, and 1% hypoxanthine aminopterin thymidine supplement (HAT, which gave a final concentration of 13.6 mg/l hypoxanthine, 0.178 mg/l aminopterin, and 3.88 mg/l thymidine) and were cultured in an incubator containing 5% CO2 at 37 C. Cells were seeded onto 35-mm culture dishes at least 2d prior to electrophysiological experiments.