8 mm pore size polycarbonate membranes

Migration assays for transfected cultured cells were carried out using 8-mm pore size polycarbonate membranes (Millipore, Billerica, MA, USA) and 24-well plates. Freshly trypsinized and washed cells were suspended in serum-free medium, and cells (200 μL, 5 × 10⁴ cells/well) were placed in the top chamber of each insert; medium (600 μL) containing 10% FBS was added into the lower chambers. After incubating for 24 h at 37 °C in a 5% CO₂ humidified incubator, cells were fixed and stained with crystal violet solution. Cells in the inner chamber were removed using a cotton swab, and cells attached to the bottom side of the membrane were counted and imaged under an inverted microscope (Olympus Corp., Shinjuku, Tokyo, Japan) at 200× magnification over 10 random fields in each well [54 (link)].

The migration assay for transfected cultured cells was carried out using 8-mm pore size polycarbonate membranes (Millipore, Billerica, MA, USA) and 24-well plates. Freshly trypsinized and washed cells were suspended in serum-free medium, and cells (200 mL, 5 × 10⁴ cells/well) were placed in the top chamber of each insert; medium (600 mL) containing 10% FBS was added into the lower chambers. After incubating for 24 h at 37 °C in a 5% CO₂ humidified incubator, cells were fixed and stained with hematoxylin. Cells in the inner chamber were removed with the use of a cotton swab, and cells attached to the bottom side of the membrane were counted and imaged under an inverted microscope (Olympus Corp., Shinjuku, Tokyo, Japan) at 200 × magnification over ten random fields in each well^{42 (link)}.