Abstract
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 μm 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.
Keywords
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Article info
Publication history
Accepted:
June 30,
2004
Received in revised form:
March 30,
2004
Received:
December 17,
2003
Identification
Copyright
© 2004 Elsevier Ireland Ltd. Published by Elsevier Inc. All rights reserved.
