Cell Adhesion Flipping Assay

Cell–cell adhesion flipping assays were performed as previously described^{44 (link),45 (link)}. Briefly, a suspension of fluorescently labeled cells in PBS⁺⁺ was sedimented for 10 min in a chamber slide containing unlabeled confluent monolayers of adherent cells. A picture was taken before flipping the dish. Slides were then filled and carefully dipped in a large vessel containing pre-warmed PBS⁺⁺. They were then rotated by 180° and maintained in an upside-down position for 15 min, allowing cells that did not adhere to the monolayer to detach. The chamber slide was then rotated back 180° and carefully removed from the large vessel. A second picture was taken after flipping, around the same position. Finally, the percentage of cells in suspension adhering to the monolayer of adherent cells was calculated. For this assay, wide-field images were rapidly acquired on a Zeiss Axio Observer 7 epifluorescence microscope equipped with a EC Plan-Neofluar 5× NA 0.16 dry objective and an ORCA-Flash 4.0 LT sCMOS camera (Hamamatsu).