Incucyte zoom 2018a software

Cells were seeded at a density of 40,000 cells/well in 12-well plates. Cell growth was monitored every 2 h by the IncuCyte Zoom live-cell analysis system (Essen BioScience Ltd.). Cell density was quantified by IncuCyte Zoom 2018A software (Essen BioScience Ltd.).

Pericytes were seeded at 8000 cells/mL into two 6‐well cell culture plate (Corning Inc., Corning, NY, USA) precoated with gelatin (Cell Biologics Inc.) in either Dulbecco's modified Eagle media (DMEM) with 4.5 g·L⁻¹ glucose, l‐glutamine, and sodium pyruvate (Corning Inc.) supplemented with +5% FBS (Corning Inc.) +1% penicillin/streptomycin (P/S) (Corning Inc.) or endothelial growth medium 2 (EGM‐2; Cell Biologics, Inc., Chicago, IL, USA) supplemented with the ECs medium supplement kit (Catalog M1168, Cell Biologics Inc., Chicago, IL, USA). Cells were seeded at 10 000 cells per well into a 24‐well cell culture plate (Corning) precoated with gelatin (Cell Biologics Inc.) in cell culture media. After 6 h for cells to attach, media was changed to include LDL (Sigma) or d‐glucose (Sigma). Plates were placed into an Incucyte Zoom instrument (EssenBioscience Inc., Ann Arbor, MI, USA) integrated with the incucyte zoom 2018A software (EssenBioscience Inc., Ann Arbor, MI, USA), and growth was monitored for 5 days to construct proliferation curves.

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.