For spontaneous migration assay, 1000 cells were seeded into wells of 96-well IncuCyte ImageLock plates. Migration assay was performed using IncuCyte® Live Cell Analysis Imaging System (Essen BioScience, Ltd., Royston, UK) for 72 h with images taken every 2 h. Using Manual Tracking plug-in (ImageJ, version 1.52p, F. Cordelieres, Institute Curie, Paris, France), cellular trajectory, velocity, and distance covered by a single cell were determined. End-point directionality was calculated as the ratio between straight-line displacement (distance to origin, DTO) and total path traveled by the cell (total distance, TD) [62 (link)]. For each parameters, 30 cells (10 cells per clone) per group were analyzed.
For collective migration assay, cells were cultured in 96-well IncuCyte® ImageLock plates. Upon reaching the confluence by the cells, wounds in the cells’ monolayers were done using IncuCyte® WoundMaker. Then, the closure of the wound was followed using IncuCyte® Live Cell Analysis Imaging System. Images collected every 2 h for 48 h were used for data analysis (IncuCyte® ZOOM 2018A software, Essen BioScience, Ltd., Royston, UK), which was presented as a percent of scratch overgrown by the cells over time.
For collective migration assay, cells were cultured in 96-well IncuCyte® ImageLock plates. Upon reaching the confluence by the cells, wounds in the cells’ monolayers were done using IncuCyte® WoundMaker. Then, the closure of the wound was followed using IncuCyte® Live Cell Analysis Imaging System. Images collected every 2 h for 48 h were used for data analysis (IncuCyte® ZOOM 2018A software, Essen BioScience, Ltd., Royston, UK), which was presented as a percent of scratch overgrown by the cells over time.
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