xCELLigence real-time cell migration assay

Migration of HUVECs transduced with shNT or shProtrudin lentiviruses was measured using an xCELLigence RTCA DP instrument (ACEA Biosciences) equipped with CIM-plate 16 [27]. Prior to the xCELLigence measurements, cells were starved for 2 h in ECM2 media containing 0.1% BSA. After the starvation period cells were trypsinized and seeded at a density of 3 × 10⁴ cells/well into upper chamber of CIM-plate 16 containing starvation media. The wells of the lower chamber were loaded with ECGM2 media with double supplements and 50 ng/mL VEGF. As the cells migrated towards the chemoattractant across microelectronics sensors integrated at the bottom side of upper chamber, the impedance was measured for 18 h at 15-min intervals. The measurements were recorded and analyzed using Real-Time Cell Analysis (RTCA) Software 1.2 (ACEA Biosciences, San Diego, CA).