Transwell cell culture chambers with 8 m pore filters

To evaluate the cell migration and invasion, Transwell assays were conducted using Transwell cell culture chambers with 8 µm pore filters (Corning Life Sciences). After treatment with 0, 10, 20, 40 µM of UA for 24 h, HCT116 and HCT-8 cells were harvested and resuspended in serum-free RPMI-1640 without UA. Then, ~5×10⁴ cells that survived after the indicated concentrations of UA treatment for 24 h were seeded into the upper chambers. The lower chambers were filled with RPMI-1640 media containing 10% FBS as a chemoattractant. Cells were allowed to migrate towards the complete medium for 12 h in the migration assay, the non-migrating cells in the upper chamber were wiped and the migrated cells were stained with crystal violet for 15 min at room temperature. For quantification, the average number of migrated cells per field was assessed by counting three random fields under a phase contrast microscope (Leica Microsystems GmbH) at a magnification of ×200. The cell invasion assay was similar to the migration assay, except that the upper chambers were coated with Matrigel matrix (BD Biosciences).

Migration assays were performed using Transwell cell culture chambers with 8-µm pore filters (Corning Life Sciences). Following the transfection of miR-155-5p mimic or inhibitor (25, 50 or 100 nM) for 48 h, A549 cells were trypsinised and resuspended in a serum-free medium. A total of 5×10⁴ cells in 200 µl of serum-free RPMI-1640 were plated in the upper chambers. RPMI-1640 medium containing 10% (v/v) FBS was used in the lower chambers as a chemoattractant. The cells were allowed to migrate for 12 h in a 37°C humidified incubator, following which the non-migrated cells were removed from the upper surface of the Transwell membrane using a cotton swab. Membranes were then stained with 0.01% crystal violet. For quantification, the average number of migrating cells per field was assessed by counting three random fields under a Leica phase-contrast microscope at a magnification of ×100. For the cell invasion assay, after transfection with 50 nM of miR-155-5p mimic or inhibitor for 48 h, similar procedure as in the migration assay was used; however, the upper chambers were coated with Matrigel matrix (BD Biosciences), and cell invasion was allowed to proceed for 24 h.