Matrigel membrane filter

Cell invasion assays were performed using modified Boyden chambers consisting of Transwell with pre-coated Matrigel membrane filter inserts in 24-well tissue culture plates (BD Biosciences) as described previously [66 (link)]. Serum-deprived cells suspended in DMEM containing 0.1% fetal bovine serum (FBS) were added to each Transwell. After incubation at 37°C for 8 h, non-invading cells were removed by wiping the upper side of the membrane, and the invading cells were fixed with methanol and stained with Diff-Quick Kit (Sysmex, Kobe, Japan). The invading cells on the filter were counted from 8 randomly selected high-power microscopic fields. The mean number of cells and standard deviations were calculated.

Cell migration assays were performed using modified Boyden Chambers (Transwells, Corning Incorporated, NY, USA) containing uncoated Transwell polycarbonate membrane fliters with 8-μm pores in 24-well plates. SiHa and CaSki cells were transfected with 50 nm miRNA or 50 nm siRNA, respectively. After 24 h, cell viability was confirmed by Trypan blue assay prior to apply for the migration assay. Then, cells with similar viability in each group were added to the upper chamber of each migration well, and 500 μl of DMEM with 10% FBS was added to the lower part of the chamber and allowed to migrate for 16 h. After gentle removal of the non-migratory cells from the filter surface of the upper chamber, cells that migrated to the lower side were fixed and stained with 1% crystal violet solution for 1 h. Then, the number of the cells migrating to the lower surface was determined microscopically by counting 5 random visual fields per well. Cell invasion assays were carried out using the chambers containing transwell precoated Matrigel membrane filter inserts with 8-μm pores in the 24-well plates at 1 × 10⁵ cells per well (BD Biosciences, USA). All assays were performed three independent times.