Assay for theTransendothelial Migration of Human Natural Killer Cells

Abstract

The natural killer (NK) cell has been shown to be important in mounting a host’s initial response to many types of invaders, such as certain types of viruses, bacteria, and protozoa, and to be involved in the control of neoplastic growth and in allograft rejection (1,2). However, before any leukocyte can perform its function in any tissue, it must first traverse the barrier of endothelial cells lining the blood vessel wall. This chapter describes a method with which to study this transmigration and the surface molecules of the NK cell that might be involved. Unlike monocytes, neutrophils, or T cells, NK cells comprise a very small percentage of peripheral blood leukocytes, only 10% of the lymphocyte fraction. This makes it a challenge to measure their transmigration, as transmigration assays usually necessitate a large number of a pure population of cells. The method described here uses a population of cells enriched for, but not exclusively, NK cells, and measures the NK cell transmigration using phenotypic analysis of the starting and transmigrating populations. The technique involves culturing endothelial cells in tissue culture well inserts that contain pores through which the NK cells can fall after their transmigration across the endothelial cell monolayer. The NK cells are enriched using negative selection exclusively, so as not to activate them by engaging their cell surface molecules, which could potentially change their pattern and extent of transmigration. For these experiments, we use the operational definition of NK cells as CD56⁺/ CD3^- cells (1,3).