Egf 50 ng ml

The organoid culture was performed as described previously (Karthaus et al., 2014 (link)). Briefly, dissociated prostate cells from 8-12 week-old C57B1/6 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 resuspended in growth factor reduced matrigel (Corning, Corning, NY) and plated in 96-well plates.

Organoid assay was performed as has been described previously.^{18 (link)} FACS-isolated basal cells, Sca-1⁺ luminal cell, eYFP⁺ Sca-1⁻ luminal cells, and eYFP⁻ Sca-1⁻ luminal cells from Pbsn-eYFP mice or FACS-isolated Sca-1⁻ and Sca-1⁺ luminal cells from WT-Sox2 and K8-Sox2 mice were cultured in Corning Matrigel Growth Factor Reduced Basement Membrane Matrix (Corning, Tewksbury, Massachusetts) with advanced DMEM/F12 supplemented with B27 (Life Technologies, Grand Island, New York), 10 mM HEPES, glutamax (Life Technologies), penicillin/streptomycin, and the following growth factors: EGF 50 ng/mL (Peprotech, Rocky Hill, New Jersey), 500 ng/mL recombinant R-spondin1 (Peprotech), 100 ng/mL recombinant Noggin (Peprotech), 200 nM of TGF-β/Alk inhibitor A83–01 (Tocris, Ellisville, Missouri), and 10 μm Y-27632 (Tocris). Dihydrotestosterone (Sigma) was added to a final concentration of 1 nM.
To collect organoids for histological and IHC analyses, 1 mg/mL dispase (Invitrogen) was added and incubated for 1 hour at 37°C. Organoids were then fixed using 10% formalin for 10 minutes and resuspended in 50 μL of Richard-Allan Scientific HistoGel Specimen Processing Gel (Thermo Fisher Scientific) for preparation of paraffin embedded blocks.