Matrigel coated 8.0 μm filter invasion chambers

Cell invasion was investigated using Matrigel-coated 8.0-μm filter invasion chambers (BD Biosciences, San Jose, CA, USA). Cells were incubated for 22 hours at 37°C in a humidified atmosphere of 5% CO₂. Cells on the upper surface of the membrane were removed using cotton tips after the indicated incubation times. The migrant cells attached to the lower surface were stained with crystal violet (500μL of 5 mg/mL crystal violet dissolved in 20% methanol) and incubated for 30 minutes. The membrane was washed with phosphate-buffered saline, and the cells on the filter were counted under an optical microscope (magnification, 200×).

Matrigel-coated 8.0-μm filter invasion chambers (BD Biosciences, U.S.A.) were used to determine the cell invasion of shCtrl or shRab7a MDA-MB-231 cells. After incubating at 37°C with 5% CO₂ for 24 h, cells were removed from the upper surface of the membrane by cotton tips. The migrant cells attached to the lower surface were stained with Crystal Violet for 30 min. The membranes were washed with PBS, and then the attached cells on the filter were detected using an optical microscope (magnification: 200×).