In Vitro prostate colony- and sphere-forming assays.
In Vitro prostate colony- and sphere-forming assays
1. Prostates were dissected, minced into small pieces with a steel blade, and digested with 0.8 mg/ml collagenase in 10 ml of primary cell medium/10% FBS at 37°C for 90 min.
2. Cells were passed through 100-μm nylon mesh (Becton Dickinson), washed twice with 20 ml of primary cell medium/10% FBS, resuspended in 1 ml of primary cell medium/10% FBS, and counted.
3. Prostate cells were dissociated by mincing and collagenase digestion, and UGSM was harvested from embryonic day 16 fetuses.
4. Colony assays were based on protocols in primary cells (5 × 104) were plated in primary cell system in each well of a six-well plate and irradiated with 500 rad the next morning.
5. Prostate cell samples were counted by hemocytometer and plated in PrEGM on top of irradiated cells.
6. Colonies were counted on days 8–10.
7. In some experiments, plates were fixed with acetone for 2 min, washed with 1× PBS, and stained with trypan blue for 1 h.
8. Prostate sphere growth and passaging conditions were based on protocols in refs.
9. Each sample of prostate cells was counted by hemocytometer, mixed with 1 × 104 cells, and suspended in 1:1 Matrigel/PrEGM in a total volume of 80 μl.
10. Each sample was plated around the rim of a well of a 12-well plate and allowed to solidify for 15 min before 2 ml of PrEGM was added.
11. Spheres were counted 7–10 days after plating.
12. For passaging of spheres, media was aspirated and Matrigel was digested by incubation in 500 μl of dispase for 30 min at 37°C.
13. Digested cultures were pelleted and incubated in 1 ml of PrEGM containing 10% collagenase for 30 min at 37°C.
14. Samples again were pelleted and incubated in 0.05% Trypsin/EDTA for 10 min at room temperature, passed several times through a 27-gauge syringe, and passed over a 40-μm filter.
15. Cells were counted by hemocytometer and replated at a density of 10,000 cells per well after each passage.
Reference
Xin L , Ide H , Kim Y , Dubey P , Witte ON PNAS (2003) 100(Suppl 1):11896–11903.
Devon A. Lawson, Li Xin, Rita U. Lukacs, Donghui Cheng , and Owen N. Witte. (2007) PNAS 104: 181-186
