Transwell Invasion Assay for Cell Migration

The Transwell invasion assay was performed using 24-well Transwell plates with polycarbonate membrane and 8.0 μm pores (Corning, USA). After 24 h of transfection, Matrigel (4.0 μg/μl, 60 μl) was added into Transwell chambers (Corning, NY, USA) and cultured for 2–3 h at 37°C for gel solidification. RPMI 1640 medium containing 10% FBS was added to the lower chambers and cells were seeded in the upper chambers with serum-free medium at a density of 4 × 10³ cells/per well. After 48 h, the migrated cells were fixed with 4% paraformaldehyde and stained with 0.1% crystal violet for 30 min. Migrating cell numbers were counted in five random fields under an inverted phase microscope (Nikon, Japan) at a magnification of 100× as previously described [13 (link)]. Assays were performed in triplicate followed by statistical analysis.