Prior to cell seeding, nanoridges or nanosawteeth surfaces were coated with 20 μg/mL collagen IV (BD Biosciences) at 4 °C for about 1 h and then mounted back to a homemade 6-well plate. Cells were trypsinized and resuspended to 1 × 104 cell/mL in each own growth medium, and then 2 mL of cell suspension was added to each well. After the cells were allowed to adhere for approximately 1 h, nonadherent cells were washed off and each well was refilled with 2 mL of fresh medium. The cells were then cultured overnight. Two hours before imaging, fresh growth medium was replaced for each cell line. For time-lapse imaging, the plate was placed in the incubator chamber (37 °C, 5% CO2) of a Zeiss Observer 2.1 microscope with an automated stage. Phase-contrast images were taken every 3 min for approximately 20 h.
Live-Cell Actin Fluorescent Imaging. Similar to individual cell migration experiments, a nanosawtooth surface was coated with 20 μg/mL collagen IV at 4 °C for about 1 h, then mounted in a homemade 35 mm Petri dish. Lifeact-eGFP/MDA-MB-231 and Lifeact-eGFP/M4 cells were harvested following the same protocol as in the individual cell migration experiments. On the second day, cells were imaged with a Zeiss LSM 880 laser-scanning confocal microscope. Both fluorescent and bright-field images were taken every 10 s using an oil-immersion, 63× objective and a zoom-in factor of 2.5.
Live-Cell Actin Fluorescent Imaging. Similar to individual cell migration experiments, a nanosawtooth surface was coated with 20 μg/mL collagen IV at 4 °C for about 1 h, then mounted in a homemade 35 mm Petri dish. Lifeact-eGFP/MDA-MB-231 and Lifeact-eGFP/M4 cells were harvested following the same protocol as in the individual cell migration experiments. On the second day, cells were imaged with a Zeiss LSM 880 laser-scanning confocal microscope. Both fluorescent and bright-field images were taken every 10 s using an oil-immersion, 63× objective and a zoom-in factor of 2.5.