Transendothelial Migration Assay for Tumor Cells

Primary CD31⁺ endothelial cells isolated from lungs by using CD31 beads (Miltenyi Biotec) were used for the transendothelial migration assay as previously described^{42 (link)}. In brief, CD31⁺ endothelial cells were cultured for 5–6 days and then seeded on gelatin coated insert in EBM-2 (Lonza). After 2 days, cell culture media was changed with DMEM plus 5% FBS in the “bottom” wells and with DMEM plus 1% FBS in the “insert” wells, followed by the treatment of vehicle or PK68 for 2 h prior to addition of RFP-LL/2 tumor cells. After 20 h, the remaining cells in the “insert” wells were removed. Then fluorescence picture of transmigrated RFP-LL/2 cells were taken at ×40 magnification under the microscope (Nikon), and the intensity of fluorescence was analyzed by Image J software.