Analysis of leukocyte migration through monolayers of cultured endothelial cells

In this chapter methods are described for analyzing the adhesion and migration of isolated leukocytes on endothelial cell monolayers that have been cultured on different substrates and treated with cytokines. When endothelial cells are grown on porous filters inserted in wells, the levels of leukocyte adhesion and migration are calculated from the number added and the numbers retrieved from the upper and lower chambers. Fluorescence microscopic examination of the fixed filters can be used to ascertain whether leukocytes are retained above or below the filter. Direct observations of the time course of migration can be made when endothelial cells are cultured in six-well plates after leukocytes are allowed to settle onto them for a short period. In a more specialized assay, leukocytes are perfused through glass capillaries coated with endothelial cells, and again, direct video-microscopic observations are made. In this assay all stages of capture, immobilization, and migration can be followed. In general, the filter-based assay has the highest throughput and greatest ease of use but yields less detailed information, whereas the flow-based assay is most difficult to set up but is most physiologically relevant.