Transwell chambers (8 μm pore size; BD Biosciences, Franklin Lake, NJ) were utilized to perform in vitro invasion assays. First, upper chambers were coated with Matrigel (R&D Systems), and 2 × 104 cells suspended in serum free medium were seeded onto the Matrigel‐coated upper chambers and 1 ml medium containing 10% FBS was placed in the lower chambers. After 20 h of incubation, upper chambers were swabbed with a cotton swab, fixed with methanol, and then stained with Giemsa solution (Sigma‐Aldrich, St. Louis, MO). The cells attached to the lower surface of the chambers were counted under a light microscope. All experiments were assayed in triplicate.
For single‐cell tracking migration assays (Hsu et al, 2018 (link)), cells were seeded into CellCarrier‐96 Ultra Microplates (1,000 cells/well) (#6055302, PerkinElmer, Waltham, MA) and incubated overnight. Then, cells were stained with Cyto‐ID Red (ENZO Life Sciences, Plymouth Meeting, PA) and the cell movements were monitored at 30 min intervals using a high content screening system (Molecular Devices, Sunnyvale, CA). Video images were collected and stored as image stacks using MetaMorph software (Molecular Devices).
For single‐cell tracking migration assays (Hsu et al, 2018 (link)), cells were seeded into CellCarrier‐96 Ultra Microplates (1,000 cells/well) (#6055302, PerkinElmer, Waltham, MA) and incubated overnight. Then, cells were stained with Cyto‐ID Red (ENZO Life Sciences, Plymouth Meeting, PA) and the cell movements were monitored at 30 min intervals using a high content screening system (Molecular Devices, Sunnyvale, CA). Video images were collected and stored as image stacks using MetaMorph software (Molecular Devices).