Measurement of the Levels of Polymerized Actin (F-Actin) in Chemokine-Stimulated Lymphocytes and GFP

The development of both microscopic and flow cytometry techniques has allowed investigators to examine the individual behavior of migratory cells under many different conditions, such as inflammation, invasion and metastasis, normal recirculation of blood cells, and so forth. Cells belonging to the immune system provide a typical example of cells in which migration is a critical feature for them to exert their physiological function (1 –3 ). Leukocyte migration requires the reorganization of the cytoskeleton of the cell to allow the transition from a medium of high flow stress, such as the blood, into the target tissues across narrow gaps provided by endothelial cell-cell junctions (4 ). Thus, cytoskeletal plasticity is a key requirement for leukocyte migration and its associated morphological changes (5 ). Such a property is provided by the actomyosin system, a flexible compartment of the cell cytoskeleton that is dynamically regulated by a number of signaling intermediates and adaptor molecules that ensure its proper polymerization/depolymerization and contractility (6 –9 ). These molecules are regulated by extracellular cues, which function as molecular on/off devices, providing the cell with the appropriate signal that triggers changes in the cytoskeleton by the recruitment and/or activation of the adequate intracellular components. Chemoattractants are among the most widely studied of these signals. They can be simple molecules, such as fMLP, or complex polypeptides, such as chemokines.