Purification and Characterization of Antigen-Binding Virgin and Memory B Cells

This chapter focuses on the methods for purifying resting antigen-specific B cells so that their activation by antigens and T cells can be assessed. Many methods for studying the physiology and activation of B lymphocytes are available. The chapter describes a method for purifying a population of TNP-specific B cells (TNP-ABC) from spleens of normal or primed mice. These cells are suitable for the analysis of early activation events of B cells in response to antigen. However, to interpret such data, an accurate measurement of the purity of the enriched population is required. The antigen-binding capacity of these cells as measured by rosetting or binding fluorescent antigen gives a far higher estimate of purity than functional assays, such as proliferation, plaque formation, and an enumeration of clones in the splenic focus assay.