8 μm pore size chamber inserts

The Transwell cell migration and invasion assays were performed using a 24-well plate with 8-μm-pore size chamber inserts (Corning, USA). The invasion assay was performed by the same procedure as the migration assay, except that the membrane was coated with Matrigel to form a matrix barrier. For the migration assay and invasion assay, 1 × 10⁵ cells were seeded in the upper chamber (BD Bioscience) in culture medium with 1% FBS, and 500 µl of culture medium with 10% FBS was added in each lower chamber, and then they were all incubated for 24 or 48 h at 37 °C. After incubation, the bottom of transwell inserts was fixed with cold methanol and stained with 0.5% crystal violet. Subsequently, migrated, and invaded cells were counted in five randomly selected fields under a microscope.

Cell migration and invasion assays were performed using a 24-well plate with 8-μm pore size chamber inserts (Corning). For migration assays, 5×10⁴ cells were placed into the upper chamber per well with the non-coated membrane. For invasion assays, 1×10⁵ cells were placed into the upper chamber per well with the Matrigel-coated membrane which was diluted with serum-free culture medium. In both assays, Cells were suspended in 200 μl of DMEM without FBS when they were seeded into the upper chamber. In the lower chamber, 800 μl of DMEM supplemented with 10% FBS was added. After incubation for 16 h at 37°C and 5% CO₂, the membrane inserts were removed from the plate, and non-invading cells were removed from the upper surface of the membrane. Cells that moved to the bottom surface of the chamber were fixed with 100% methanol for 20 min and stained with 0.1% crystal violet for 30 min. Then, the cells were imaged and counted in at least 10 random fields using a CKX41 inverted microscope (Olympus, Japan). The assays were conducted three independent times.

Plates with 8 μm-pore size chamber inserts (Corning, USA) were used for transwell assay. The chamber was coated with Matrigel, then seeded 2 × 10⁴ cells into the upper chamber. Cells were suspended in 200 μl RPMI 1640 medium with 0.1 % FBS in the upper chamber. In the lower chamber, 800 μl of RPMI 1640 medium was supplemented with 10 % FBS. After incubating at 37 °C for 24–48 h, some cells would migrate and invade through the Matrigel-coated membrane, attaching to the lower side of the membrane. Cells were fixed with 4 % paraformaldehyde for 10 min and stained with 0.1 % crystal violet for 25 min. Then, striking off upper cells that attached to the membrane’s inner side, invasive cells were imaged by CKX41 inverted microscope (Olympus, Japan) and counted by image J (ImageJ, RRID: SCR_003070).

Cell migration and invasion assays were performed in 24-well plates with 8-μm-pore size chamber inserts (Corning, USA). For the migration assays, 5 × 10⁴ cells in 200 μL of serum-free culture medium were seeded into each well of the upper chamber with the noncoated membrane, and 800 μL of medium supplemented with 10% FBS was added to the lower chamber. For invasion assays, 1 × 10⁵ cells in 200 μL of serum-free culture medium were seeded into each well of the upper chamber with the Matrigel-coated membrane, while 800 μL of medium supplemented with 10% FBS was added to the lower chamber. Cells that migrated through the membrane were fixed with 100% methanol, stained with 0.1% crystal violet for 30 min, imaged, and counted under a light microscope (Olympus, Japan).

Matrigel invasion assays were performed in 24-well plates with 8 μm-pore size chamber inserts (Corning, New York, USA). Upper chambers were precoated with 60 μL of Matrigel. After 30 min coating with Matrigel, 2 × 10⁴ cells in 200 μL of serum-free culture medium were seeded into each well of the upper chamber, and 500 μL of medium supplemented with 10% fetal bovine serum (FBS) were added to the lower chamber. Chambers were incubated at 37 °C with 5% CO₂ for 72 h, and then cells that migrated through the membrane were fixed with 4% paraformaldehyde, stained with 0.1% crystal violet for 30 min, and imaged and counted under a light microscope (Olympus, Tokyo, Japan).

Migration and invasion tests were conducted based on previous research with a few modifications [22 ]. Utilizing a 24-well plate with 8 μm pore size chamber inserts (Corning, USA), migration and invasion of tumor cells were evaluated. In the upper chamber, 1 × 10⁵ cells were seeded for the migration assay. The membrane was coated with Matrigel (BD Biosciences, USA) to form a matrix barrier for the invasion assay, and then 1 × 10⁵ cells were added to the upper chamber. Each lower chamber received 600 μl of DMEM medium containing 10% FBS. At 37 °C, cells were allowed to migrate for 24 h or invade for 36 h. After incubation, the cells that had migrated through the pore were fixed with 4% paraformaldehyde and stained with 0.1% crystal violet. The cells were then counted and photographed using an inverted IX71 microscope (Olympus, Tokyo, Japan).