Uplanfl 10 0.3 objective

The microfluidic device is made of PDMS (RTV615, NY, United States) by soft lithography from a patterned SU-8 silicon wafer. Glass coverslips were plasma bounded to PDMS layer. Each device consists of 10 channels of 50 μm in wide and height, 3 mm in length. In migration assay, all channels were coated with 10 μg/ml fibronectin for 1 h at 37°C. Cells were incubated with Hoechst 33342 for 10 min and subsequently washed twice with PBS and replaced with complete DMEM. Cells were collected from culture dishes using trypsin-EDTA, and resuspended in complete DMEM to a concentration of 2 × 10⁷ cells/ml. Twenty microliters of cell suspension was added to the device inlet. Cells were allowed to adhere and spread overnight. All wells of the device were then filled with 120 ml of complete DMEM. Devices were incubated at 37°C and 5% CO₂ before imaging by Olympus IX73 inverted microscopy with the UplanFL 10 × /0.3 objective (Olympus, Tokyo, Japan). Average migration velocity and directional migration duration were quantified by tracking the nucleus movement in between 5 min imaging cycles for 10 h. Only cells that did not collide with one another were selected for measurements.

Cells were seeded in a 6-well cell culture plate with a cell density of 25,000/cm² and cultivated at 37°C in 5% CO₂ overnight. Subsequently, the cell monolayers were scratched with a sterile 0.2 mL pipette tip to create linear wounds and washed with PBS to remove detached cells. Cells were incubated in a serum-free DMEM medium to eliminate cell proliferation and then observed by Olympus IX73 inverted microscopy with the UplanFL 10×/0.3 objective (Olympus, Tokyo, Japan). The migration rates were measured using ImageJ.