Matrigel pre coated transwell chambers

Transwell assays were performed according to the manufacturer’s protocol (BD Biosciences, Bedford, MA, USA) using Transwell chambers (for migration assays) or Matrigel pre-coated Transwell chambers (for invasion assays). Forty-eight hours after transfection, cells were collected, and these cells were resuspended (2×10⁴ cells per well) into 200 μL of serum-free medium and plated in the upper chamber. Add 500 μL of RPMI 1640 with 20% FBS to the lower compartment. After 36 hrs of incubation, the upper chamber membrane was fixed and stained. An inverted microscope was used to count invading cells.

Migration assay was performed using Transwell chamber (BD Biosciences, San Jose, CA, USA). Matrigel pre-coated Transwell chambers (BD Biosciences) were used for invasion assays. Briefly, 5×10⁴ cells were resuspended in FBS-free medium in strict accordance with the manufacturer’s instructions, and then subsequently placed in the Transwell chamber. Medium containing 10% FBS was added to the lower chamber. After culturing for an additional 24 hrs, cells in the upper chamber were wiped off, and cells in the lower chamber were fixed in 4% paraformaldehyde and then stained using 0.1% crystal violet.

For transwell invasion assay, transfected cells were suspended in FBS-free DMEM medium. In total, 200 µL of cell suspension containing 5×10⁴ cells was placed into the upper compartments of the Matrigel pre-coated transwell chambers (BD Biosciences, Franklin Lakes, NJ, USA), followed by the addition of 600 μL DMEM supplemented with 20% FBS in the lower compartments. After being cultured for 24 h, the non-invasive cells remaining on the upper side of the Transwell filter were gently wiped away with cotton wool. The invasive cells were fixed with 100% methanol, stained with 0.5% crystal violet, and then, washed with phosphate buffer solution. The invasive ability was determined by counting the number of invasive cells in five randomly selected microscopic fields using an inverted light microscope (IX71; Olympus Corporation, Tokyo, Japan). In the transwell migration assay, the Matrigel was not used to precoat the transwell chambers. The remaining experimental procedures were similar to those used for the transwell invasion assay.