4.7. Cell Migration and Cell Invasion Assays by Boyden Chamber Assay

For the cell migration assay, at 48 h post-transfection, 1 × 10⁵ transfected cells were resuspended in 200 µL serum-free DMEM medium and placed in the upper chamber with polycarbonate membrane pore size 8.0 µm (REF353097, Falcon, Monfalcone, Italy) placed in a 24-well plate (SKU: 353047, Falcon), with the lower chamber containing 700 µL of 10% FBS DMEM medium as a chemoattractant, and incubated at 37 °C with 5% CO₂ overnight. Non-migratory cells on top of the membrane were removed using cotton-tipped applicators. Then, migratory cells were fixed with 70% ethanol for 10 min, stained with 0.2% crystal violet solution for 10 min and washed gently. Migratory cells were counted under an inverted microscope.

The cell invasion assay was performed by using the transwell chamber coated with 35 µL of ECM at a concentration of 250 µg/mL and incubated at 37 °C with 5% CO₂ for 2 h. Then, 1 × 10⁵ transfected cells were resuspended in 200 µL serum-free DMEM medium and placed in the chamber coated with ECM gel placed in a 24-well plate (SKU: 353047, Falcon). Next, 700 µL of 10% FBS DMEM medium as a chemoattractant was added in the lower chamber and incubated at 37 °C with 5% CO₂ overnight. Non-invasive cells on top of the membrane were removed using cotton-tipped applicators. The invasive cells were fixed with 70% ethanol for 10 min, stained with 0.2% crystal violet solution for 10 min and washed gently. Invasive cells were counted under an inverted microscope. The percentages of migration and invasion were calculated; cells transfected with non-targeting siRNAs (NTC) served as a negative control.