OBJECTIVE: To visualize and quantitatively analyze spatiotemporal dynamics of individual living monocytes during transendothelial migration (TEM). METHODS AND RESULTS: We developed an in vitro new experimental system using confocal laser scanning microscope with following two improvements: (1) ultra thin collagen gel layer (30-50 microm thick) constructed under human umbilical vein endothelial cell layer for three-dimensional observation with high magnification; (2) appropriate fluorescent labeling of living monocytes and endothelial cells to keep highest cell activity. Individual monocytes behaved quite diversely. Approximately 70% of adhered monocytes directionally crawled to intercellular junction, and started invasion. Time from adhesion to start of invasion was 8.6 +/- 5.4 min (mean +/- S.D., n=61 monocytes). Approximately 80% of such invading monocytes completed TEM, but remaining 20% of once invading monocytes hesitated transmigration, and returned onto the endothelial surface. Time from start to finish of invasion was 6.3 +/- 3.2 min (mean +/- S.D., n=53 monocytes). CONCLUSIONS: Using our collagen gel-based newly-developed system, we visualized and quantitatively analyzed detailed spatiotemporal, three-dimensional dynamics of individual living monocytes during TEM. We revealed that monocytes encountered at least two hurdles, at starting invasion, and leaving endothelium, to achieve complete TEM. Approximately 56% (80% of 70% of adhered monocytes) passed both hurdles.
OBJECTIVE: To visualize and quantitatively analyze spatiotemporal dynamics of individual living monocytes during transendothelial migration (TEM). METHODS AND RESULTS: We developed an in vitro new experimental system using confocal laser scanning microscope with following two improvements: (1) ultra thin collagen gel layer (30-50 microm thick) constructed under human umbilical vein endothelial cell layer for three-dimensional observation with high magnification; (2) appropriate fluorescent labeling of living monocytes and endothelial cells to keep highest cell activity. Individual monocytes behaved quite diversely. Approximately 70% of adhered monocytes directionally crawled to intercellular junction, and started invasion. Time from adhesion to start of invasion was 8.6 +/- 5.4 min (mean +/- S.D., n=61 monocytes). Approximately 80% of such invading monocytes completed TEM, but remaining 20% of once invading monocytes hesitated transmigration, and returned onto the endothelial surface. Time from start to finish of invasion was 6.3 +/- 3.2 min (mean +/- S.D., n=53 monocytes). CONCLUSIONS: Using our collagen gel-based newly-developed system, we visualized and quantitatively analyzed detailed spatiotemporal, three-dimensional dynamics of individual living monocytes during TEM. We revealed that monocytes encountered at least two hurdles, at starting invasion, and leaving endothelium, to achieve complete TEM. Approximately 56% (80% of 70% of adhered monocytes) passed both hurdles.