Transwell Migration Assay for Exosome-Induced Cell Movement

Migration was determined using 8μ-pore filters for the Transwell migration assay (BD Biosciences), according to the manufacturer's instructions and as previously described [63 (link)]. Briefly, murine Raw264.7 cells and human pre-osteoclast cells (1×10⁵ cells/well) treated for 24hrs with MM cell-derived exosomes (25μg/ml) were washed and resuspended in DMEM high glucose medium supplemented with 1% of ultracentifuged FBS. Cells were then placed in the upper migration chambers, whereas the lower chamber contained DMEM high glucose medium supplemented with 10% ultracentifugated FBS. Filters were removed after 24 hours, fixed in methanol and cells stained with Diff-Quick (Medion Diagnostics GmbH, Düdingen, Switzerland). Alternatively, pre-osteoclast cells were not treated and MM cell-derived exosomes (25μg/ml) were added directly in the lower chamber contained DMEM high glucose medium supplemented with 10% ultracentifugated FBS. Two independent experiments were performed in triplicate; cells from 5 different fields were counted for each condition.