Migration chambers with an 8 μm pore size membrane

Migration and invasion assays were performed using migration chambers with an 8‐μm pore size membrane (Corning, Corning, NY, USA). Prior to invasion assay, 60 μL of DMEM with 2% Matrigel (BD Biosciences, San Jose, CA, USA) was added to an upper chamber. Cells (2 × 10⁴ for migration assay or 2 × 10⁵ for invasion assay) were resuspended in serum‐free DMEM and added to the upper chamber. DMEM with 10% FBS was added to lower chamber to allow cells to migrate or invade for 24 or 36 h, respectively. Migrated or invaded cells on the lower surface of the chamber membrane were fixed with 4% paraformaldehyde and then stained with 5% crystal violet (Sigma‐Aldrich, St Louis, MO, USA). Each experiment was performed in triplicate, and mean values of migrated or invaded cells were calculated.

Transwell migration assay was performed by using migration chambers with an 8-μm pore size membrane (Corning, Corning, NY, USA). Matrigel invasion assay was performed according to the BioCoat Matrigel Invasion Chamber system (BD Biosciences, San Jose, CA, USA). Cells (2 × 10⁴) were re-suspended in serum-free DMEM and added to an upper chamber. Lower chambers were loaded with 10% fetal bovine serum and cells were allowed to migrate or invade for 24 h under 1 or 21% O₂. Migrated or invaded cells on the lower surface of the membrane were fixed with 100% methanol and then stained with 5% crystal violet (Sigma-Aldrich, St Louis, MO, USA). The number of migrated or invaded cells per field was counted under a phase contrast microscope. The mean±s.d. was calculated from the number of six different fields.