Transfected PC3 or DU145 cells were cultured in 6.5-mm transwell inserts with an 8 μm pore size, coated with either collagen I or 0.5 mg/μL of growth factor-reduced Matrigel, purchased from Corning Life Science (Acton, MA, USA). Cells were seeded into the upper chambers in 100 μL of fetal bovine serum (FBS)-free medium, while 10% FBS-containing medium was placed in the lower chambers as a chemo-attractant, and incubated in either 1% O2 or 21% O2 for 24 h. Cells on the lower surface of the transwell inserts were fixed in 4% formaldehyde and stained with hematoxylin and eosin, and four high-power independent fields of each membrane were counted.
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