Measurement of the motility of endothelial cells in confluent monolayers

Objective: The reported experiments were designed to develop and test a method to measure the motility of endothelial cells in confluent cultures. Methods: Endothelial cell cultures were recorded by time-lapse video. Frames from the recording were digitized, and cell locations were identified at 30-minute intervals using either the centroids or nuclei. The speed of the cells was determined for distances between their successive positions. Cells were stained immunocytochemically to determine the status of their adherens junctions. Results: Prior to reaching confluence cells exhibited motility unhampered by collisions. After reaching confluence, cells continued to exhibit motility that decreased with time until the cells ceased effective movement. The motility in confluent monolayers persisted in the presence of complete adherens junctions. Motility could be restored in quiescent confluent cultures by the addition of fibroblast growth factor 2 (FGF-2). Generally, cells responding to FGF-2 moved without obvious loss of adherens junctions. Conclusion: These experiments provide evidence that (1) endothelial cells in culture are motile, (2) they do not form circumferential adherens junctions until the culture reaches confluency, (3) the establishment of adherens junctions itself does not interdict cell motion, and (4) the immotile state is reversible by FGF-2 in the presence of adherens junctions.