Transwell cell migration assay kit

Cell migration ability was measured using a Transwell cell migration assay kit (BD Biosciences, Franklin Lakes, NJ, USA). Cells were harvest during the logarithmic growth phase and single cell suspension was prepared with a cell density of 5×10⁴ cells per 1 ml RPMI-1640 medium. Subsequently, 0.1 ml cell suspension containing 5×10³ cells was added into the upper chamber, RPMI-1640 (Thermo Fisher Scientific, Inc.) supplemented with 20% fetal calf serum (Sigma-Aldrich; Merck KGaA) was used to fill the lower chamber. After incubation for 24 h, membranes were collected and subjected to staining with 0.5% crystal violet (Sigma-Aldrich; Merck KGaA) for 20 min at room temperature. Membranes were observed under an optical microscope (Olympus Corporation, Tokyo, Japan) and the stained cells were counted. The upper chamber was pre-coated with Matrigel (cat. no. 356234; Millipore, USA) before the cell invasion assay, all other steps were the same as for the migration assay. Cell migration and invasion were normalized to the group with the largest number of migrating and invading cells, which was set to 100.

A Transwell cell migration assay kit (BD Biosciences, Franklin Lakes, NJ, USA) was used to evaluate cell migration ability. Briefly, cells were collected and centrifuged at 600 × g for 5 min at room temperature and were used to prepare a cell suspension with a density of 5×10⁴ cells per well, and 100 µl cell serum-free suspension containing 5×10³ cells was added to the upper chamber. Then, RPMI-1640 medium (Thermo Fisher Scientific, Inc.) containing 20% fetal calf serum (Sigma-Aldrich; Merck KGaA) was added into the lower chamber. Following incubation at 37°C with 5% CO₂ for 24 h, membranes were collected and stained with 0.5% crystal violet (Sigma-Aldrich; Merck KGaA) at room temperature for 15 min. Stained cells were counted under an optical microscope (Olympus Corporation, Tokyo, Japan) at magnification, ×20. A cell invasion assay was performed using the same protocol, but the upper chamber was pre-coated with Matrigel (EMD Millipore, Billerica, MA, USA) at 4°C for 4 h.