Boyden chamber migration and invasion assays.

Cells (5 × 10⁴) were resuspended in serum-free medium, seeded in a transwell insert (for migration) or a Matrigel-coated transwell insert (for invasion) in the presence of cell culture medium with 10% FBS at the bottom chamber, and exposed to 20% or 1% O₂ for 16 hours (migration) or 24 hours (invasion). Cells that invaded to the lower side of the transwell insert were fixed with methanol, stained with 0.5% crystal violet, and counted.