Transwell cell migration plates

For the wound healing test, different groups of 1 × 10⁶ RKO, HCT116 and SW480 cells, and cells were cultured to near 90% confluence in the plates. Then scratched with a sterile 1 ml pipette tip. After the damaged cell layer was washed, it was cultured in complete medium without FBS. The wounds were photographed with a light microscope at 0 and 24 h.
Transwell cell migration plates (Corning Incorporated) were used for invasion and migration assays. Transwell migration experiments were carried out using Corning 8‐μm chambers according to the manufacturer's instructions (BD Biosciences). For the invasion assay, cancer cells (5 × 10⁴ cells/well) from different groups (RKO/Control; RKO/BMAL1;RKO/shControl; RKO/shBMAL1; SW480/Control; SW480/BMAL1; SW480/shControl; SW480/shBMAL1) with 200 μl serum‐free medium were placed into the upper chamber, with 600 μl medium supplemented with 10% FBS in the bottom chamber. After 36 h, the migrated cells in the underside of the membrance were immersed and washed with PBS, fixed with 4% paraformaldehyde, stained using 0.1% crystal violet, washed three times with water, and counted under light microscopy.

Cell migration and invasion were determined using transwell cell migration plates (Corning, NY, USA) and Matrigel invasion chambers (Matrigel-coated membrane, BD Biosciences, San Jose, CA, USA) as previously described [19] . Briefly, the cells (1.0×10⁴) were seeded in serum-free medium into the upper chamber and allowed to invade toward 10% FCS in the lower chamber as a chemoattractant. After 12 h (for migration assays without matrigel coating) or 48 h (for invasion assays with matrigel coating), the cells that had invaded through the membrane and adhered to the underside of the membrane were counted as previously described [16] (link).