Transwell upper chamber inserts

The 24-well culture plate, including transwell upper chamber inserts, was used for transwell invasion assays (Corning, New York, USA). First, 1 × 104 cells or cells transfected with OE-RNA and/or control vectors were mixed with 100 ml serum-free RPMI-1640 or DMEM with different concentrations of ibuprofen (C643 at 0, 0.5, 1, and 1.5 mM, and OCUT-2C at 0, 1, 2, and 3 mM) added to the upper chamber. Then, 600 ml of medium containing 10% FBS with the same concentrations of ibuprofen was added to the lower chamber. After culture at 5% CO₂ at 37 °C for 48 h, the chamber was removed from the plates and fixed with 4% paraformaldehyde for 30 min. The cells that traversed through the membrane pores were stained with crystal violet. Ultimately, the number of cells passing from the upper chamber to the lower chamber was observed through an inverted microscope (Olympus, Tokyo, Japan).

The transwell invasion assay was performed with 24-well culture plates and by matching transwell upper chamber inserts (Corning, New York, USA). Briefly, 2 × 10⁴ cells were mixed with 200 μL serum-free RPMI-1640 medium, and were seeded in the upper chamber inserts pre-coated with Matrigel matrix (BD, New Jersey, USA). Then, 600 μL medium containing 10% FBS was added to the lower chamber. After incubating at 37°C for 24 h, the cells on the upper membrane surface were wiped off using a cotton swab, and the cells that had traversed the membrane were stained with crystal violet. Finally, the cells were counted using an inverted microscope (Olympus, Tokyo, Japan), and the images were evaluated using the ImageJ software.