Tissue Digestion and Cell Isolation Protocol

Fresh tissue samples were transported in ice-cold saline and immediately dissected into portions for fixation in 10% formalin followed by paraffin embedding. For human specimens, a 4-hour enzymatic digestion into single cells was performed at 37°C in 35 mL Hanks’ balanced salt solution (HBSS) containing 5 mg/mL Collagenase Type I (Life Technologies), 10 μM ROCK Inhibitor Y-27632 (StemRD), 1 nM dihydrotestosterone (DHT) (Sigma-Aldrich), 1 mg DNAse I (Roche), and 1% antibiotic/antimycotic solution (100×; Corning).^{4 (link)} Mouse specimens were digested for 1 hour in HBSS containing either 10 mg/mL cold protease or 1.5 to 2 mg/mL Collagenase Type I, plus 10 μM ROCK Inhibitor Y-27632, 1 nM DHT, 1 mg DNAse I, and 1% antibiotic/antimycotic solution.