Cyto idtm red

To examine the 3D cell migration capability, cells were labeled with Cyto-IDTM Red (ENZO Life Sciences; Cat# ENZ-51037-K025) and seeded to 96-well culture plates that had been pre-coated with Matrigel (Corning; Cat# 356231). Cell motility was recorded every hour for 24 consecutive hours under a Leica DMI6000B fluorescence microscope (Leica Microsystems) and further analyzed with the MetaMorph 7.7.5 software (Molecular Devices).

For invasion assays, transwell chambers (8 μm; Falcon, NJ, USA) were coated with Matrigel (30 μg; BD Biosciences, Bedford, MA, USA). For both migration assays and invasion assays, the plates were seeded with 10⁵ cells per well and incubated for 20 h. Cells that invaded or migrated from the top to the bottom of the chamber were fixed in methanol and stained with 50 μg ml⁻¹ propidium iodide. The cells were subsequently photographed under a fluorescence microscope and counted using AIS software (Imaging Research Inc., St Catherine's, Canada). Each condition was assayed in triplicate.
3D invasion assays were performed by first pre-coating 96-well culture plates with Matrigel (BD Biosciences) and incubating overnight at 37 °C to allow Matrigel polymerization. Cells (5 × 10³), labelled with Cyto-IDTM Red (ENZO Life Sciences, Plymouth Meeting, PA, USA), were seeded on Matrigel-coated plates, and cell invasion ability was observed under a Leica DMI6000B fluorescence microscope (Leica Microsystems GmBH, Wetzlar, Germany) for 24 h. The velocity and motility tracts of cell migration were analysed and quantified using MetaMorph 7.7.5 software.