Purification and Characterization of Murine MZ and T2-MZP Cells

The spleen is the second major reservoir of B cells in the adult. In the spleen, cells, generated in the bone marrow, are selected, mature, and become part of the peripheral B-cell pool. Murine spleen comprises several B-cell subsets representing various maturation stages and/or cell functions. The spleen is a complex lymphoid organ organized into two main structures with different functions: the red and white pulp. The red pulp is flowed with blood while the white pulp is organized in primary follicles, with a B-cell area composed of follicular B cells and a T-cell area surrounding a periarterial lymphatic sheath. The frontier between the red and white pulp is defined as the marginal zone (MZ) and contains the MZ B cells. Because B cells, localized in different areas, are characterized by distinct expression levels of B-cell receptor (BCR) and of other surface markers, splenic B-cell subsets can be easily identified and purified by flow cytometry analyses and fluorescence-activated cell sorting (FACS).Here, we will focus on MZ B cells and on their precursors, giving some experimental hints to identify, generate, and isolate these cells. We will combine the use of FACS analysis and confocal microscopy to visualize MZ B cells in cell suspensions and in tissue sections, respectively. We will also give some clues to analyze B-cell repertoire on isolated MZ-B cells.