Abstract
Leucocyte transendothelial migration (TEM) or diapedesis is pivotal in leucocyte trafficking during the inflammatory and immune responses. The endothelium plays an active role in this process, triggering an array of signalling pathways and reorganizing its cytoskeleton and membrane to facilitate leucocyte TEM. Diapedesis can occur between endothelial cells (paracellular) or through individual endothelial cells (transcellular). This latter route accounts for up to 30% of the total diapedesis in certain endothelial cell types in vitro. Mechanisms underlying both routes of diapedesis have been subjected to intense investigation during recent years. Here we describe a method to discriminate between the paracellular and the transcellular routes of diapedesis in vitro. The method is based on a transmigration assay of human T lymphoblasts through TNF-α-stimulated human primary endothelial monolayers, a triple fluorescence labelling of F-actin, the adhesion receptor ICAM-1 and the junctional protein β-catenin and a subsequent acquisition of z-stacks of high-resolution confocal sections.
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Acknowledgments
E.C.M. is supported by a contract from the Spanish Juan de la Cierva program. S.G. is supported by a fellowship from the Spanish Ministry of Science and Education. J.M. was supported by a British Heart Foundation intermediate fellowship (no. FS/04/006), grant SAF2008-1936, and a contract from the Spanish Ramón y Cajal program.
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Cernuda-Morollón, E., Gharbi, S., Millán, J. (2010). Discriminating Between the Paracellular and Transcellular Routes of Diapedesis. In: Marelli-Berg, F., Nourshargh, S. (eds) T-Cell Trafficking. Methods in Molecular Biology, vol 616. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-60761-461-6_5
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DOI: https://doi.org/10.1007/978-1-60761-461-6_5
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