Culturing Murine and Human Prostate Cancer Cell Lines

Murine embryonic fibroblast 3T3-L1 cells (ATCC, #CL-173™) were maintained in a proliferative medium (PM) made up of DMEM high glucose (4.5 g/L glucose), supplemented with 10% newborn calf serum, 4 mM L-glutamine, 1 mM, sodium pyruvate and 1% antibiotic–antimycotic cocktail (100 U/mL penicillin, 10 µg/mL streptomycin, 0.25 µg/mL amphotericin B). Murine PCa cells TRAMP-C1 (ATCC, #CRL-2730™) were cultured in DMEM high glucose supplemented with 5% fetal bovine serum (FBS), 4 mM L-glutamine, 5% Nu-serum IV complement, 0.005 mg/mL insulin, 10 nM dehydroepiandrosterone (DHEA) and 1% of an antibiotic–antimycotic cocktail. Human androgen-dependent LNCaP cells (ATCC, #CRL-1740) were cultured in RPMI 1640 supplemented with 10% FBS, 2 mM L-glutamine, 15 mM HEPES, and 1% of antibiotic cocktail (100 U/mL penicillin, 10 µg/mL streptomycin). Human androgen-independent PC-3 cells (ATCC, #CRL-1435) were cultured in DMEM/F12 medium supplemented with 10% FBS, 2 mM L-glutamine, and 1% of an antibiotic–antimycotic cocktail. All cell lines were cultured at standard conditions (T^ª: 37 oC, CO₂: 5%). FBS was stripped from small weight molecules, including steroids, by using a protocol previously published [16 (link)]. Charcoal-stripped FBS (csFBS) was employed to perform a cell culture medium without androgens as a substitute of FBS.