Hct 8 cells

On day 1, 1 × 10⁴ HCT-8 cells (colon cancer cell line ordered from ATCC [Manassas, VA, USA]) were transfected with NC or siKCNJ5 and seeded in lower chamber. On day 2, 1 × 10³ M2 macrophages in 100 μL of complete DMEM were added to the upper insert (8.0 μm pore; 24-well Transwell, Corning). With incubating at 37°C and 5% CO₂ for 24 h, the non-migrated cells in the insert were wiped away, and the migrated cells were stained with DAPI for 30 min. The transwell membranes were removed and pasted on microslides. Finally, the fluorescence graphs from the slides were captured with an Upright metallurgical microscope (BX53, Olympus, Tokyo, Japan). Each experiment was conducted in triplicate. The cell chemotaxis model was constructed with FALCON (8.0 μm pore size cell chambers, 353097). The chamber loaded with HCT-8 cells was put in a corner of the lower chamber, whereas 5 × 10³ M2 macrophages were seeded in the opposite corner (Figure 7I). Cell chemotaxis potential on the flat surface was detected using this model. First, M2 macrophages were marked with the bioactive fluorescence probe, Dil (Beyotime, Shanghai, China); 24 h later, the images of chemotactic accumulation of M2 macrophages to HCT-8 cells were observed with the Upright metallurgical microscope. Cell counting was performed with ImageJ software (NIH, Bethesda, MD, USA).