24 well cell migration chambers with 8 μm pore size inserts

Colon cancer cell lines HCT8 and HCT116 were cocultured with HUVEC by using 24-well cell migration chambers with 8 μm pore size inserts (Corning). First, HCT8 and HCT116 were plated in the lower chamber and transfected with miRNAs mimic/inhibitor and (or) FOXM1 plasmid for 12 h, and then cultured in 2% FBS medium. HUVEC (1.25 × 10⁴/well) were seeded into the insert and cocultured with colon cells for 24 h. Non-migrated HUVEC were removed by cotton swabs from the upper surface of the insert and cells on the lower surface of the insert were fixed and stained. Migrated cells were photographed by digital microscope in five random fields and number of migrated cells was calculated and determined as the mean value.

Colon cancer cell migration was evaluated using 24-well cell migration chambers with 8 μm pore size inserts (Corning Coster, Corning, NY, USA). Colon cancer cells were serum-starved overnight and transfected with either mimic-miR-340-5p (50 nM), mimic-Ctrl (50 nM), TSB, and TSB-Ctrl for 24 h in Opti-MEM serum reduced media as previously described^{24 (link)}. Briefly, cells were transfected with mimic-mir340-5p, mimic-Ctrl, TSB (50 nM), and TSB-Ctrl (50 nM) for 24 h. After transfection, 1 × 10⁶ cells/ml were loaded in the inserts and DMEM with 10% serum in the lower chambers and incubated for 24 h at 37 °C (5% CO₂). Non-migrated cells were removed from the upper surface of the insert and cells on the lower surface of the insert membrane were fixed in ice-cold 100% methanol. After washing with PBS, cells were stained with 0.5% crystal violet. In separate experiments, cells were pre-incubated for 30 min with the Rho kinase inhibitor, Y-27632 (50 µM) (R&D systems Europe, Abingdon, UK). Cells were counted microscopically by using high power field (HPF) in five different fields. Data were expressed as the mean number of migrated cells per high power field.

Chemotaxis of CT-26 cancer cells in response to neutrophil co-culture was evaluated using 24-well cell migration chambers with 8 μm pore size inserts (Corning Coster, Corning, NY, USA). Colon cancer cells were serum starved overnight and resuspended in serum-free DMEM with 0.5% BSA and co-cultured with neutrophils in 2:1 neutrophils/cancer ratio and incubated with different concentrations of DNase I and loaded in the inserts. DMEM with or without 200 ng CXCL2 (Peprotech, Rocky Hill, NJ, USA) was added in the lower chambers and incubated for 24 h at 37°C (5% CO₂). Non-migrated cells were removed by cotton swabs from the upper surface of the insert and cells on the lower surface of the insert membrane were fixed in ice-cold 100% methanol and stained with 1% crystal violet. All migrated cells were counted microscopically in at least 5 different fields. Migration index was then calculated as the ratio of the number of migrated cells divided by the number of cells in the control wells.