DMEM/F-12

Protocol tips

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Defrost the growth factor reduced Matrigel in advance, on ice or in fridge

Upstream tips
Defrost the growth factor reduced Matrigel in advance, on ice or in fridge

Publication protocol

Prostate organoid culture and prostate regeneration assay: The organoid culture was performed following the previous study [18]. Briefly, dissociated prostate cells from 8–12 week-old C57Bl/6 mice were cultured in DMEM/F12 supplemented with B27 (Life technologies, Grand Island, NY), 10 mM HEPES, Glutamax (Life technologies, Grand Island, NY), Penicillin/Streptomycin, and the following growth factors: EGF 50 ng/ml (Peprotech, Rocky Hill, NJ), 500 ng/ml recombinant R-spondin1 (Peprotech, Rocky Hill, NJ), 100 ng/ml recombinant Noggin (Peprotech, Rocky Hill, NJ), 200 µM TGF-β/Alk inhibitor A83-01 (Tocris, Ellisville, MO), and 10 µM Y-27632 (Tocris, Ellisville, MO). Dihydrotestosterone (Sigma, St. Louis, MO) was added at 1 nM final concentration. Cells were mixed with growth factor reduced matrigel (Corning, Corning, NY) by 1:1 ratio and plated in 96-well plates. In some experiments, enzalutamide (Selleckchem, Houston, TX) was added to a final concentration of 10 µM. To collect prostate organoids, culture media was removed and 100 µl of 1 mg/ml Dispase solution (Invitrogen, Carlsbad, CA) was added and incubated for 1 hour at 37°C. Samples were then transferred to 1.6 ml Eppendorf tubes and centrifuged at 850×g for 2 minutes. Organoids were fixed with 10% formalin for 10 minutes and resuspended in 100 µl of HistoGel (Richard-Allan Scientific, Kalamazoo, MI) for preparation of paraffin embedded blocks. To dissociate prostate organoids, organoids released from matrigel were resuspended in 300 µl of chilled Trypsin-Versene (Lonza, Walkersville, MD) and were gently passed through 28-gauge insulin syringes approximately 10 times for 5 minutes. It usually took an extra two minutes to dissociate Type III organoids due to their bigger sizes. Subsequently, 200 µl of organoid culture media was added and centrifuged at 850×g for 2 minutes. Dissociated cells were resuspended in culture media. Viable single cells were enumerated under a microscope and plated in 96-well plates with growth factor reduced matrigel (Corning, Corning, NY).

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