Trypsin edta

Sphere formation assays were performed as previously described (17 (link)). In brief, cells were seeded in 24-well ultra-low attachment plates at 1 x 10³ cells/well in 500 µl of MammoCult® Human Medium Kit (Catalog # 05620, Stem Cell Technologies, Vancouver, Canada). Spheres were counted between 7–10 days after plating, using Gel Count TM—Oxford OPTRONIX version 1.03. To subculture the tumorspheres for secondary and tertiary generation, supernatant was removed and 1 mL of pre-warmed Trypsin-EDTA (0.25%) (Stem Cell Technologies) was used to dissociate the tumorspheres. Pellets were resuspended in MammoCult Human Medium and viable cells were counted according to the manufacturer’s instructions. Similar cell densities were plated to form subsequent generations.

Mouse prostate tissues were dissected and fixed in 4% paraformaldehyde for subsequent cryo-embedding in OCT compound (Sakura), or fixed in 10% formalin followed by paraffin embedding. For dissociation, prostate tissues were dissected and minced to small clumps, followed by enzymatic dissociation with 0.2% Collagenase/Hyaluronidase (StemCell Technologies) in DMEM/F12 media with 5% FBS for 3 h at 37°C. Tissues were digested with 0.25% Trypsin-EDTA (StemCell Technologies) for 1 h at 4°C, passed through 21- to 26-gauge syringes and filtered through a 40-mm cell strainer to obtain single-cell suspensions. Dissociated prostate cells were suspended in Hanks’ Balanced Salt Solution Modified/2% FBS.

For isolation of YFP-lineage marked cells, the bladder urothelium was manually dissected from the underlying connective tissue and the isolated urothelial tissue was digested at 37°C for 3 hours in 1x collagenase/hyaluronidase (10x stock, Stem Cell Technologies, #7912, Cambridge, MA) in DMEM-F12 and 5% fetal bovine serum. Samples were pelleted at 350 X g for 5 min at 4°C in an Eppendorf 5810R tabletop centrifuge, re-suspended in 0.25% trypsin/EDTA (Stem Cell Technologies, Cambridge, MA), and incubated for 0.5 hour on ice. Cells were collected by centrifugation as above, and incubated in a cocktail of pre-warmed dispase (5 U/ml) plus DNase I (1 mg/ml stock, final concentration 0.1 mg/ml) (Stem Cell technologies, Cambridge, MA) for 1–2 minutes with vigorous pipetting. Following which, cells were filtered using Corning™ Falcon™ Test Tube with Cell Strainer Snap Cap (#08–771-23), pelleted and re-suspended in Hanks′ Balanced Salt solution (HBSS, Stem Cell Technologies, Cambridge, MA) plus 2% fetal bovine serum (FBS, Gibco Laboratories, Gaithersburg, MD) for sorting using a BD FACS Aria II sorter. The YFP-expressing cells were isolated using PE/FITC (R-Phycoerythrin/Fluorescein isothiocyanate) channels to gate the YFP positive (YFP+) population.