Time-lapse imaging of YTS and 721.221 cells was performed as described previously (64). YTS cells were labeled with 5 μM CellTrace Violet, whereas 721.221 cells were labeled with 0.5 μM Calcein Green (both from Thermo Fisher Scientific). YTS and 721.221 cells were mixed at a 1:1 ratio and suspended 4:1 with cold growth factor–reduced Matrigel membrane matrix (Corning) containing 1 μM TO-PRO-3 (Thermo Fisher Scientific) to detect dead cells. The Matrigel was solidified in chambered coverglass for 30 min at 37°C before imaging. Time-lapse images of cells were taken every 3 min over 8 hours at 37°C, 5% CO2 on a confocal microscope equipped with a White Light Laser and a 20× 0.75 NA objective (TCS SP8, Leica Microsystems). Images were analyzed with the TrackMate Plugin (65) for ImageJ and visually scored for contact formation.

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