Chapter 15 - Identification and Isolation of Human Splenic Macrophages, Lymphocytes, and Related Cells with in Situ Immunohistochemical Techniques and Countercurrent Centrifugal Elutriation

Abstract

This chapter discusses a technique for identification and isolation of spleen macrophages, lymphocytes and related cells. The analysis of tissue sections for various types of cells offers a number of advantages over the use of cell suspensions for this purpose: (1) the identification of the cell type of interest in situ allows quantitative estimates of the number of such cells per unit of tissue, (2) the application of sensitive detection techniques in situ provides a way of determining whether subsequent manipulations have altered cellular phenotype, (3) the microanatomic distribution of cells expressing a particular phenotype, especially in complex tissues. In an experiment described in the chapter, the presence of various cell membrane antigens on spleen cells was determined using monoclonal antibodies and heteroantisera together with the avidin-biotinylated horseradish peroxidase and the avidin-biotinylated glucose oxidase techniques. Elutriation was performed at 25°C. The system was sterilized by pumping 70% ethanol through the lines and by then flushing with sterile elutriation medium. The ability to obtain large numbers of splenic microphages in this manner is particularly important for experiments in which minority subpopulations are being studied.