Matrigel-based Cell Invasion Assay

The cell invasion assay was performed as previously described [35 (link),36 (link),37 (link)]. Briefly, 5% Matrigel (Becton Dickinson Biosciences, Bedford, MA, USA) was used to coat the membrane of the upper insert of a Millicell invasion chamber (Millipore, Burlington, MA, USA) with a pore size of 8 μm in a 24-well plate. Cells in a medium containing 1% FBS were seeded into the upper insert. The lower chamber contained a medium with 20% FBS to trap invading cells. After 16 to 24 h of incubation at 37 °C, the invasive cells were determined by observing the reverse side of the upper insert after being fixed with formaldehyde and stained with crystal violet.