24 well transwell migration chambers

Transwell migration and invasion assays were carried out using 24-well transwell migration chambers (Corning Life Sciences, NY, USA) with 8-μm pore size polyethylene membranes. For the migration assay, cells were placed in the upper chamber of each insert without Martigel coating. For the invasion assay, cells were placed in the upper chamber of each insert, which was precoated with Matrigel (BD Biosciences, USA). For both assays, RCC cells were starved overnight and trypsinized and suspended in serum-free medium. The 1×10⁴ cells (for the migration assay and invasion assay) were seeded in starvation medium on the top chamber. The bottom chamber was filled with 10%FBS in medium which acted as chemoattractant. After 24h incubation, the cells migrating or invading to the lower chamber were fixed with anhydrous ethanol for 30min, stained with crystal violet solution for 30min, and counted from 10 randomly chosen fields using a microscope. Three independent experiments were performed, and the data are presented as the average±SD.

After transfection with si-NC or si-CNTFR, melanoma cell lines were seeded into a 6-well plate and then scratched with a 200 μl pipette tip when the degree of cell fusion approaches 90%. After incubation with a serum-free medium for 24 h, the width of the wounds was examined under a microscope.
Moreover, the migratory ability of cells was assessed by transwell migration assay using the 24-well transwell migration chambers (8-mm pore size; Corning, NY, United States). In short, cells with a density of 5×10⁴/ml were resuspended in 200-ml serum-free DMEM and then plated into the inner chambers (Omar Zaki et al., 2019 (link)). Similarly, a 500 µl DMEM medium containing 20% serum was added to the bottom chambers as an attractant. After 24 h of incubation, cells on the transparent membrane were washed with PBS, fixed with 4% paraformaldehyde for 30 min, and counterstained with 0.1% crystal violet. Finally, the number of migrated cells was calculated by ImageJ software.

Migration assays were performed using 24-well transwell migration chambers (Corning, Corning, New York, USA) with polyethylene membranes (8 μm pore size). The upper chambers were seeded with 2.5 × 10⁴ cells/well in 200 μl of serum-free DMEM supplemented with 0.1% BSA. The cells were allowed to migrate for 10 h at 37 °C. In co-culture migration assays, 3 × 10⁴ LN229 cells/well were seeded in lower chamber 12 h before experiments. Afterward, cells at the upper layer of the membrane were scraped and cells at the lower layer were stained with Giemsa solution and photographed under a microscope. The number of cells was quantified in randomly selected fields. Three independent experiments were performed.