HTR8/SVneo cell migration was determined by wound healing assays and performed in siC and siD7 as previously described [15 (link)]. To perform cell migration assay in stable shC and shD7 cells, they were seeded on twelve-well plates (1 × 105 cells/well) and cultured to reach a confluence of 70%. Then, cells were transfected or not with EV-GFP, p-Cx43-GFP, EV, D7.I, or D7.II plasmids for 48 h, as indicated. Confluent monolayers were wounded and assessed under microscopy at 0 and 8 h after. The results were expressed as the percentage of the wound closure calculated as the difference between the remaining area at 8 h and the initial one at 0 h recorded in seven fields of duplicate wells from three independent experiments.
Analysis of the migration of individual cells was conducted employing an IN Cell Analyzer 2500 HS (High content analysis, GE Healthcare Life Sciences) in a time-lapse mode (one picture every 15 min interval over a total time of 120 min). Individual cell trajectories were manually tracked using Image J/ Fiji software. Only cells at the front line of migration were examined, using the border of the cell as the reference mark for cell movement. Movies are presented in theS1 –S3 Movies.
Analysis of the migration of individual cells was conducted employing an IN Cell Analyzer 2500 HS (High content analysis, GE Healthcare Life Sciences) in a time-lapse mode (one picture every 15 min interval over a total time of 120 min). Individual cell trajectories were manually tracked using Image J/ Fiji software. Only cells at the front line of migration were examined, using the border of the cell as the reference mark for cell movement. Movies are presented in the
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