Abstract
Methods are described for analysing adhesion and migration of isolated lymphocytes on endothelial cell monolayers which have been co-cultured with different stromal cells, with or without additional cytokine treatment. The different cells types are grown on opposite sides of 3.0- or 0.4-mum pore filters depending on whether migration through the whole construct is to be analysed or adhesion to the endothelial cells alone. Assays may be "static" or filters can be incorporated into flow chambers so that cell behaviour can be directly observed under conditions simulating those in vivo. In general, by choice of method, one can evaluate efficiency of attachment and ability of cells to migrate across the endothelial monolayer, through the filter and through the stromal cell layer. Fluorescence microscopic examination of fixed filters can be used, e.g. to ascertain whether lymphocytes are retained by stromal cells. In general, static assays have the higher throughput and greatest ease of use, while the flow-based assays are more physiologically relevant and allow detailed recording of cell behaviour in real time.