Quantitative Wound Healing and Cell Migration Assays

Wound healing and Transwell assays were performed as previously described [4 (link), 11 (link)]. Briefly, after appropriate treatments, cells were seeded in 6-well plates and cultured until they reached 90% confluence. A 10-μl micropipette tip was used to make a wound. Cells were monitored at 0 h and 48 h after scratching and images of wound healing were captured (magnification of 100×) using a inverted phase contrast light microscope (Olympus, Tokyo, Japan) with DP Controller software (Olympus Life Science, Tokyo, Japan). Cell migration was quantified by measuring the wound healing index; i.e., the wound area healed by the cells at 48 h after scratching relative to the wound area at 0 h, using ImageJ software. For the Transwell migration or invasion assays, cells were resuspended in DMEM without serum and seeded into the upper chamber of 8-μm Transwell filters (Merck Millipore, Berlin, Germany). The invasion assay was performed using filters pre-coated in 1:3 diluted matrigel (BD Biosciences, Bedford, MA, USA) while the migration assay was not. DMEM containing 15% FBS was added to the lower chambers (24-well plate) and the cells were incubated 16 h for the migration assay and 24 h for the invasion assay. The invaded or migrated cells were quantified after 0.1% crystal violet staining in five randomly selected fields (magnification of 200×).