Establishing 3D Cell Cultures for Longitudinal Imaging

A visual inspection was carried out after 24 h to identify plates with consistent confluence across wells. Cells were washed in PBS, trypsinized as described above and resuspended in PC3 medium. Typically, one-half to two-thirds of cells from each well were used for subsequent 3D cultures depending upon the initial confluence and effectiveness of trypsinization. Medium was supplemented with 2% Growth Factor Reduced Matrigel (GFRM; BD Biosciences) and added to 96-well ImageLock plates (Satorius) pre-coated with 10 μl of GFRM for 15 min at 37°C. For live imaging, ImageLock plates were incubated at 37°C for 4 h, then imaged using an IncuCyte ZOOM (Satorius) with IncuCyte ZOOM Live Cell Analysis System Software 2018A. Phase and GFP images were taken every hour for 4 d at two positions per well with ×10 objective lens.