Functional Assays by Flow Cytometry

Cell function assays have been redefined over the past several years, largely because of technologies such as flow cytometry. The major difference between other techniques and those proposed using flow cytometry is the ability to analyze viable, single cells in a rapid and quantitative fashion. One of the advantages of flow cytometry methods lies in the ability to obtain population information. This means that what may appear to be a homogeneous population using conventional bulk assays may be demonstrated to be multiple populations by flow cytometry. This chapter deals primarily with phagocytes-macrophages and polymorphonuclear cells. Many factors can cause alterations in either the number or function of phagocytic cells. Among these factors are a variety of drug interactions. For example, corticosteroids commonly cause a significant increase in the number of circulating leukocytes (primarily the result of accelerated neutrophil release from bone mar row stores), together with a decrease in neutrophil adher ence to vascular endothelium, migration of cells out of the vasculature, and a slight increase in the neutrophil circulating half-life. Neutrophils are normally stored in the bone marrow for 5 to 7 days before being released into the blood. Granulocyte colony-stimulating factor and interleukin 1 play key roles in this release of neutrophils from the marrow. Neutrophils in the circulating pool have a half-life of about 7 h, after which they marginate and emigrate through tissue, where they remain functional for 1 to 2 days. The removal of neutrophils, irrespective of their state of activation, is primarily via phagocytosis by macrophages or by disposal through mucosal surfaces. Because neutrophils are shortlived reactive cells present in very high numbers, minor alterations in their function can have profound effects in microenvironments. The purpose of this chapter is to define some of the procedures that can be used to evaluate the function of neutrophils or macrophages by flow cytometry.