Isolation and Characterization of Prostate Stem Cells

This process was a modification of a well-established protocol^{12 (link), 39 (link)}. Briefly, minced prostate tissues were digested in 1 mg/mL collagenase (Sigma-Aldrich) in RPMI-1640 (Gibco) media containing 10% FBS (Corning) with shaking at 37°C for 2 hours, followed by trypsinization. Dissociated cells were passed through 20G needles and 40 μm cell strainers to eliminate aggregates, followed by removal of red blood cells by ACK buffer. To enrich murine bPSC, isolated cells were stained with Zombie Violet Live/Dead Fixable Viability Dye (Biolegend) in PBS, followed by incubation with 1:100 diluted fluorescence-conjugated specific antibodies (Biolegend): CD45-FITC (#103108), CD31-FITC (#102506), Sca-1-APC (#122512) and CD49f-PE (#313612). Human prostate samples from unidentified patients were acquired from Indiana University School of Medicine Tissue Repository under IRB-approved protocols. Isolated human prostate cells were stained with Zombie UV Fixable Viability Dye (Biolegend), CD45-FITC (#304006), EpCAM-PE (#324206), CD26-APC (#302710) and CD49f-BV421 (#313624) as described in detail by Strand et al.^{40 (link)}. Once stained, fluorescence activated cell sorting was performed on the BD FACSAria under sterile conditions.