RPMI 1640, with L-Glutamine and 25mM HEPES

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	supplemented with 10% fetal calf serum (FCS) (PAA), 2mM L-glutamine (Life Technologies), and antibiotics (100 U/mL of streptomycin and penicillin).

Protocol tips
supplemented with 10% fetal calf serum (FCS) (PAA), 2mM L-glutamine (Life Technologies), and antibiotics (100 U/mL of streptomycin and penicillin).

Publication protocol

Human PCa cell lines LNCaP, PC3, DU145, VCaP, and CWR22RV1 were obtained from ATCC. DUCaP and BPH-1 were a generous gift from Dr Schalken (Center for Molecular Life Science, Nijmegen, The Netherlands), PC3-AR and LAPC-4 from Dr Cato (Karlsruhe Institute of Technology, Karlsruhe, Germany), and RWPE-1 from Dr Watson (Conway Institute, Dublin, Ireland). EP156T were generated by immortalization of primary cells with human telomerase reverse transcriptase (24, 25). For routine culture, cells were maintained at 37°C in a humidified 5% CO2 atmosphere in RPMI 1640 (Lonza) supplemented with 10% fetal calf serum (FCS) (PAA), 2mM L-glutamine (Life Technologies), and antibiotics (100 U/mL of streptomycin and penicillin). LNCaP cells required, in addition, 2.5 g/L of D-glucose (Invitrogen), 10mM HEPES, and 1mM Na-pyruvate (Lonza). PC3-AR cells were cultured in the presence of geneticin (G418) (500 μg/mL; Life Technologies) to preserve the expression of AR, whereas 100nM dihydrotestosterone was added to the culture medium of LAPC-4 cells. VCaP cells were kept in DMEM low-glucose medium (Fisher), supplemented with 10% FCS, 2mM L-glutamine (Life Technologies), and 1.75 g/L of D-glucose (Invitrogen). EP156T and RWPE-1 cell lines were cultured as recommended (24, 25). Generally, for treatment with the synthetic androgen methyltrienolone (R1881) (1nM) or the antiandrogen enzalutamide (MDV3100) (10μM) cells were seeded in RPMI 1640 supplemented with 10% charcoal/dextran-treated FCS (Fischer) for 2 days before incubation with the indicated reagents for 24 and 48 hours, or as stated. Cell pellets were collected at the mentioned time point and frozen at −20°C for future use. Reagents and R1881 were purchased from Sigma-Aldrich unless otherwise specified. The antiandrogen MDV3100 was obtained from Eurasia.

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Download the product protocol from Lonza for RPMI 1640, with L-Glutamine and 25mM HEPES below.

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