Transwell Assay for Cell Migration

Cell migration was assayed using transwell polycarbonate membrane inserts (6.5 mm; Greiner Bio-One) with 8 µm pore size as described [29 (link)]. After 24 h of starvation, 5 × 10⁴ cells were plated in the top insert chamber with 200 µL serum-free DMEM. Each of the collagen matrices was placed in the low chamber with 800 µL 10% FCS/DMEM. Cells were allowed to migrate across the filter for 18 h at 37 °C before fixation in Shandon™ Formal-Fixx™ (ThermoFisher Scientific), and staining with 0.1 mg/mL crystal violet (Sigma-Aldrich Chemie GmbH, Buchs, Swtzerland) solution. Images of duplicate inserts were acquired on an Olympus CKX41 microscope equipped with a ProgResCT3 camera. Migration was quantified by using the ImageJ software as described [29 (link)]. Data represent means ± SD from three independent experiments performed with three different cell donors for each cell type.