Cell migration
A migration assay was performed using transwell 24-well Boyden chambers
(Corning, Corning, USA) with an 8.0 μm pore size membrane without
matrigel. Cells were seeded in the upper chamber at a density of
5×104cells/well in 200 μl serum-free RPMI-1640 medium.
As a chemoattractant, 600 μl 10% FBS containing complete medium was
added to the lower chamber. After incubation for 6 hours at 37°C in
humidified 5% CO2, the upper chamber was detached from
the well, and the cells at the lower chamber, representing migrated
cells, were counted using a cell counting chamber.