Fy these cells . In place of utilizing PNA (see “pitfalls”), the Fas receptor (CD95, Apo-1) is often applied to recognize GC B cells (Fig. 141B), which is hugely upregulated in these cells . With all the described four colors, CD19 or B220, CD38, GL7, and either PNA (Fig. 141A) or Fas (Fig. 141B), GC B cells can unambiguously be identified. Considering that GC B cells also downregulate IgD [1194, 1198, 1199], that is an more marker that could be added to the staining protocol. Pitfalls: 1 pitfall in the staining in Fig. 141A is that the PNA signal is downregulated really quick if the time in between staining and measurement with the samples is long (own observation). Continually keeping samples on ice nonetheless helps to counteract this downregulation. two.2.six Information analysis: Germinal Center B cell subpopulations: The GC has a specific microanatomic structure that may be divided into the DZ, exactly where B cell proliferation and somatic hypermutation take spot, along with a LZ, where selection of high-affine B cell clones happens. In an effort to stain for these two zones, GC B cells are initially stained as described inside the section “Murine Germinal Center B cells” above (Fig. 141). CD86 (also referred to as B7) is a surface protein which is expressed on activated B cells [1200, 1201] and has a costimulatory function for T cell proliferation . Together with all the chemokine receptor CXCR4, that has been shown to be crucial for GC organization , Victora et al. applied the mixture of CD86 and CXCR4 to differentiate DZ cells (CXCR4hi CD86low) from LZ cells (CXCR4low CD86hi) . The staining for DZ/LZ cells is shown in Fig. 142. Pitfalls: A pitfall of this staining is the difficulty to set an correct gate for the DZ/LZ, considering the fact that these two populations are certainly not clearly separable from one another by FCM. In particular if fluorochromes for CXCR4 and CD86 are utilized which can be Integrin alpha V beta 5 Proteins Biological Activity identified for fluorescence spillover, suitable compensation is quite crucial to not distort DZ/LZ ratios. See also Chapter II Section 1 Compensation.Author Manuscript Author Manuscript Author Manuscript Author ManuscriptEur J Immunol. Author manuscript; accessible in PMC 2020 July ten.Cossarizza et al.Page2.Human B cells and their subsetsAuthor Manuscript Author Manuscript Author Manuscript Author Manuscript2.three.1 Overview: B cells represent the Ab-producing cells building from na e B cells to Ab-secreting Computer. One feature of B cells is their capacity to differentiate upon antigen dependent and independent stimulation to Ab secreting cells, also named plasma cells. The stages of B cell differentiation share many prevalent capabilities amongst the human plus the rodent immune method. In this section, we concentrate on human B cells. 2.3.2 Introduction: To determine B cells, the B cell distinct molecules CD19 and/or CD20 serve as distinct surface Neurturin Proteins Purity & Documentation markers (Fig. 143). CD19 can be a B cell surface molecule expressed at the time of immunoglobulin heavy chain rearrangement , CD20 is expressed by all mature B cells beyond the pro B cell stage within the bone marrow and disappears on the surface of mature plasma cells [1206, 1207]. For additional discrimination of B cell developmental stages, combinations of further markers for example CD27, CD38, CD23, CD77, and expression of surface Igs are used. Immature CD19+ B cells within the bone marrow express higher levels of CD38 and variable levels of CD20 and IgM, which raise with their additional differentiation (Figs. 143F and 144) . CD38++ CD20++ immature B cells express IgM and IgD, leave the bone m.