Matrigel membrane

Cell invasion assays were performed in 24-well transwell plates (8-μm pore size, Corning Costar, USA), as described elsewhere [8 (link)]. The chamber inserts were coated with a Matrigel membrane (BD Biosciences, USA). RPMI-1640 containing 20% fetal bovine serum in the lower chamber served as the chemoattractant. The cells (5 × 10⁵/ well) were incubated at 37°C for 48 h.

1–2 × 10⁵ P3 SSEA-1⁺ or SUSD2⁺ cells were resuspended in 100 μl thawed Matrigel membrane (BD Biosciences, USA) and placed in drops on the bottom of 48-well plates. Then, the plates were inverted and placed in the 37 °C incubator for 20 min to allow gelation. After solidification of cell-Matrigel mixture, the plates were added with 1 ml/well TEM to cover the Matrigel and the TEM was changed every 3–4 days. To obtain intact spheres for passing or immunofluorescence staining, 1 ml/well precooled Cultrex Organoid Harvesting Solution (R&D Systems, USA) was added and incubated on ice for 40 min. Spheres were centrifuged at 300 g for 5 min and washed wish PBS to remove the depolymerized Matrigel. For suspension culture, 1–2 × 10⁶ P3 SSEA-1⁺ or SUSD2⁺ cells were seeded in the low-attachment 6-well plate. The SUSD2⁺ cells began aggregation within 6 h and formed a solid and non-opaque spheres after 24 h in TEM. Hollow and translucent organoids were observed in SSEA-1⁺ cells after 24 h in TEM.

For the cell invasion assay, B7-H3 KD cells and corresponding MG-63 control cells were seeded in a 24-well upper chamber coated with Matrigel membrane (BD Bioscience, USA) and cultured in serum-free DMEM medium overnight. Subsequently, 600 μl complete medium with 10% FBS was added to the lower chambers as a chemoattractant. Cells were then incubated for 24 h at 37°C, and the invasive cells attached to the lower membrane surface were stained with 0.1% crystal violet, photographed, and counted.
For the wound healing assay, B7-H3 KD cells and control cells were used at a density of 5 × 10⁵ cells/well in six-well plates. The scratched wound was created using a pipette tip and rinsed twice with phosphate buffered saline to remove free floating cells and debris. Twenty-four hours after scratching, each group of cells began to migrate into the wound surface and was visualized to evaluate the average distance of the migrating cells. All experiments were repeated three times.

Cell migration and invasion were tested by transwell insert (6.4-mm diameter and 8-μm pore size) purchased from BD Biosciences. For migration assay, the lower chamber was added with complete medium, and the upper insert was added with 5 × 10⁴ cells in serum-free medium. Cells in transwell system were cultured for another 48 h, and then subjected to crystal violet (0.25%) staining. Stained cells were observed under a microscope (× 100), and migratory cell number was counted. For invasion assay, the insert was coated with Matrigel membrane (BD Biosciences), and 5 × 10⁵ transfected cells were used.