Prostate Cell Culture Protocol

Prostate epithelial and prostate cancer cell lines were obtained from American Type Culture Collection (ATCC) (Rockville, MD), which include RWPE1 (immortalized human prostate epithelial cell line) ^{38 (link)}, and prostate cancer cell lines isolated from distinct metastatic sites from prostate cancer patients such as androgen-dependent cell line LNCaP (derived from a lymph node lesion), and androgen-independent cell lines DU145 (derived from brain) and PC3 (derived from bone) ^{39 (link)}. RWPE1 cells were maintained in keratinocyte serum free medium containing 50μg/ml gentamycin, 0.05mg/ml bovine pituitary extract (BPE), and 5ng/ml epidermal growth factor (EGF) (Invitrogen, Carlsbad, CA). LNCaP cells were maintained in RPMI-1640 medium containing 4mM L-glutamine, and 50μg/ml gentamycin. DU145 and PC3 cells were cultured in Eagles minimum essential medium with Earle’s salts with 0.1 mM of the following amino acid supplements: L-alanine, L-asparagine, L-aspartic acid, L-glutamic acid, L-proline, L-serine and L-glycine. The medium contained 4 mM L-glutamine, 2.5 g/l NaHCO₃, 1.5 mM HEPES, 100 U/ml penicillin, 100 μg/ml streptomycin, 0.25 μg/ml amphotericin B and 50 μg/ml gentamycin. MEM and RPMI media (Mediatech, Herndon, VA) were supplemented with 5% fetal bovine serum (HyClone, South Logan, Utah).