Analysis of L-selectin-mediated cellular interactions under flow conditions

Lymphocyte homing is mediated by a specific interaction between L-selectin expressed on lymphocytes and its ligands expressed on high endothelial venules (HEVs) in lymph nodes under physiological flow conditions. In this chapter, two methods for detecting L-selectin-mediated cellular interactions under shear stress mimicking physiological flow conditions are described. First, a modified Stamper-Woodruff cell-binding assay using leukocytes labeled with a fluorescent orange dye, CMTMR, is introduced. In this method, leukocytes are allowed to bind to frozen lymph node sections under shear stress and their binding to HEVs can be clearly visualized by fluorescence microscopy. Second, a parallel flow chamber assay is described. In this assay, leukocytes are allowed to roll on L-selectin ligand-expressing cells under various levels of shear stress and their adhesive interactions are recorded by a video camera equipped with an inverted microscope. These methods can be applied to determine the effects of various agents that might affect L-selectin-mediated lymphocyte homing and recruitment.