For the assessment of serum exosomes derived from OS patients on the cell proliferation of 143B and U2OS, cells were seeded at a density of 2000 cells per well in 96-well plates. The cells were treated with different exosome concentrations of 10, 20, 30, 40 μg /ml for 24, 48 and 72 h. Cell proliferation was analyzed using Cell Counting Kit 8 (Dojindo, Kumamoto, Japan) according to the manufacturer's protocol.
For the evaluation of serum exosomes of OS patients and healthy donors on the migration and invasion of 143B and U2OS, cells (6 × 104) were respectively seeded into the non-coated upper chamber of transwell plates (8 mm pore size; Corning) for a migration assay and into matrigel coated upper chamber (BD Bioscience, 354,234) for an invasion assay with 50 μg/ml exosomes. For the evaluation of exosomes derived from OS on the migration and invasion of 143B and U2OS, the same procedure as above mentioned was performed with 10 μg/ml and 50 μg/ml. After culturing for 24 h, cells were fixed with methanol and stained with a 0.1% crystal violet solution. Migrated cells were evaluated in five fields per well under a microscope.
For the evaluation of serum exosomes of OS patients and healthy donors on the migration and invasion of 143B and U2OS, cells (6 × 104) were respectively seeded into the non-coated upper chamber of transwell plates (8 mm pore size; Corning) for a migration assay and into matrigel coated upper chamber (BD Bioscience, 354,234) for an invasion assay with 50 μg/ml exosomes. For the evaluation of exosomes derived from OS on the migration and invasion of 143B and U2OS, the same procedure as above mentioned was performed with 10 μg/ml and 50 μg/ml. After culturing for 24 h, cells were fixed with methanol and stained with a 0.1% crystal violet solution. Migrated cells were evaluated in five fields per well under a microscope.