Investigating CXCR4-dependent CRC Cell Migration

CRC cells were seeded in 24-well plates and grown in culture medium. At 90% confluence, the medium was replaced with medium without FBS, containing or not containing (control) PMPs, and the cells were incubated for 4 h at 37 °C in a humidified atmosphere with 5% CO₂. To determine whether migration was CXCR4-dependent, cells were incubated with the CXCR4 antagonist AMD3100 (at a final concentration of 10 μM) or with anti-CXCR4 antibodies (at a final concentration of 100 μg/ml). SDF-1 (at a final concentration of 400 ng/ml) was added after incubation of CRC cells with PMPS to stimulate CXCR4-dependent cell migration. Cell migration was evaluated by measuring the cell-free surface at the beginning of the experiment (immediately after the scratch was made) and every 2 h until a 24-h period (Additional file 2: Supplementary Methods). The migration and invasion properties of tumour cells labelled with CellTracker after incorporation of PMPs were evaluated in uncoated or Matrigel-coated (10 mg/ml, Thermo Fisher Scientific) Boyden chambers, respectively (Additional file 2: Supplementary Methods).