Transwell chambers with polycarbonate membranes

Cell migration was measured using transwell chambers with polycarbonate membranes (Corning) as described in our previous publication [55 (link)]. Cell invasion was measured using Matrigel Basement Membrane Matrix (BD Biosciences), according to the method as described in manufacture protocol.

The migration and invasion capacity of cells was detected in vitro using Transwell chambers with polycarbonate membranes (251.87 mm in diameter, a pore size of 6 μm) (Costar Corning, USA). In the migration assay, the transfected cells were resuspended in 10 μL of serum-free medium at a density of 100 × 5/mL and added to the upper chamber. Next, 12 μL of DMEM supplemented with 10% FBS was added to the lower chamber. After incubation for 20 h, the cells on the upper membrane surface were removed, and the migrating cells were fixed with methanol and stained with 80% Giemsa stain solution. Finally, the stained cells were counted microscopically in five randomly selected fields, and the average was taken. In the invasion assay, the Transwell membranes were applied to 500 μL of Matrigel solution (37 ng/μL, BD Biosciences, USA), and the cells were incubated at 37°C for 4 h. The remaining steps were the same as those in the migration assay.

To determine the effect of each mutant on cell migration, transwell migration assay was performed by using 24-well Transwell chambers with polycarbonate membranes (8.0-μm pore size; Costar, Corning, NY, USA). Each WT- and mutant-transfected MDA-MB-231 cells were suspended in serum-free RPMI media and added to the upper compartment at 1×10⁵ cells per well. To the lower compartment, RPMI containing 5% fetal bovine serum was added. The cells were incubated for 6 h at 37 °C and in 5% CO₂ incubator. Then they were fixed with 70% methanol for 15 min, washed with phosphate-buffered saline three times, stained with hematoxylin (Sigma Aldrich) for 10 min and washed with distilled water. After removing the non-migrant cells from the top face of the membrane with a cotton swab, the membranes were excised from the chamber and mounted with Gel Mount (Biomeda, Foster City, CA, USA). The migrant cells were counted with three randomly selected scopes in high-power fields (×10).