Upper chamber

Stably transfected osteosarcoma cells in logarithmic growth were collected. For the migration assays, 100 µl of cell suspension (2×104 cells) in culture medium without FBS was inoculated into the upper chamber (Costar Inc., USA). For the invasion assays, 100 μl of Matrigel (Corning) was added at a concentration of 250 μg/ml to the upper chamber followed by incubation at 37 °C for 1 hour, and the cell suspension (4.0×104 cells) in culture medium without FBS was then added to the upper chamber. To induce cell migration, 500 μl of medium containing 20% FBS was added to the lower chamber. After MG63 and U2OS cells were incubated for 24 hours in the Migration assays and 36 hours in the invasion assays, cells in the upper chamber were wiped with cotton swabs, fixed with methanol for 20 minutes and stained with crystal violet for 30 minutes. Cells were observed and imaged using an inverted microscope (Olympus, Tokyo, Japan).

The upper chamber (8 μm pore size; Costar, Sigma-Aldrich, St. Louis, MO, USA) was coated with 30 µL Matrigel (0.32 mg/mL) and allowed to be set for 1 h. Next, a 200 µL cell suspension (5 × 10⁴ cells) in a serum-free medium containing DAPT or DMSO was added to the upper chamber (8 μm pore size; Costar). Then, a culture medium (500 μL) supplemented with 20% FBS was added to the lower chamber. After 24 h, the transwell membrane was washed twice with PBS. The non-invaded cells from the upper side of the membrane were gently removed using a cotton swab. The invaded cells on the filter membrane were fixed with cold methanol for 15 min. The invaded cells were stained using 0.1% crystal violet solution for 10 min. The stained cells from 5 random fields were photographed and counted (200× magnification). The invaded cell numbers were also measured by eluting the stain in 33% acetic acid solution, and its absorbance was measured at 570 nm [76 (link)].