Biocoat matrigel invasion chamber system

Transwell migration assay and Matrigel invasion assay were performed using migration chambers (Corning) and the BioCoat™ Matrigel™ Invasion Chamber system (BD Biosciences), respectively. Cells (2 × 10⁴) in serum-free DMEM were added to an upper chamber. In the lower chamber, 10% fetal bovine serum was used as a chemoattractant. The cells were allowed to migrate or invade for 24 h. The cells on the lower surface of the membrane were fixed with methanol and then stained with 5% crystal violet (Sigma-Aldrich). The number of migrated or invaded cells per field was counted and the mean ± SD was calculated from the number of six random fields.

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.