Live Imaging of LeukocyteEndothelium Interactions

Leukocyte extravasation is a highly dynamic, interactive, and coordinated process that plays a central role during the inflammatory response of innate immunity. The interaction of leukocytes with the activated endothelium under shear forces is comprised of many sequential events, each involving specific leukocyte and endothelial receptors, as well as chemokines and adaptor and signaling molecules. Because of its complexity, researchers studying leukocyte extravasation at the subcellular level have been forced to search for appropriate in vitro models that mimic pathophysiological conditions at sites of inflammation. We report methods for direct visualization of cellular and molecular processes of critical importance to spatiotemporally dissect the different steps in the adhesion cascade. These methodologies include techniques for the study of the dynamics of individual molecules involved in a discrete part of the process, as well as simple procedures to label molecules and cells in order to observe the extravasation process.