Cell Growth, Colony Formation Assay and Soft-Agar Colony Assay

For cell growth assay, 1 x 10 ³ /well DU145 cells were inoculated in 96-well plates. Each sample equally spread 6–8 wells. 3–4 h before measuring, each well was added with 10 μl CCK 8 solution. The absorbance was finally determined at 450 nm using a microplate reader. The absorbance values of the cells were recorded at 0, 24 h, 48 h, 72 h and 96 h, respectively. Data are expressed as mean + SD. Statistical differences between groups were analyzed by the two-way ANOVA test. P < .05 was considered statistically significant.

For colony formation assay, 2 x 10² /well DU145 cells or HeLa cells were seeded in 6-well plates. Each sample equally spread 3–4 wells. Cells were cultured in DMEM medium containing 10% FBS for 2–3 weeks. To visualize cell colonies, cells were fixed with 10% formaldehyde and stained with Giemsa stain.

For soft-agar colony assay, the method was described before [52]. This assay was performed in 6-well plates in triple with a base of 2 ml of DMEM medium containing 5% FBS with 0.6% Bacto agar (Amresco). DU145 cells were seeded at a density of 2 x10³ cells /well in 2 ml of 0.35% agar gel with 5% FBS, and were layered on the base gel. The photographs of the colonies developed in soft agar were taken and the number of colonies was scored by Photoshop about 2–3 weeks after seeding.

Data are expressed as mean + SD. Statistical differences between groups were analyzed by the two-tailed Student's t test. P < .05 was considered statistically significant.