Arge pre-B cells (pre-B II cells). Staining for additional markers such as AA.4.1, heat-stable antigen (HSA), surrogate light (SL) chains VpreB and lambda5 is often utilised to perform a much more detailed evaluation of B lineage subpopulations in BM [1113, 1114, 1121123, 1130, 1131] (Table 43). two.1.6 Data analysis: Murine B cells in secondary lymphoid organs: For identification of B cells inside the spleen and also other secondary lymphoid organs, single cells really should be gated in line with their scatter properties, and doublets should really be excluded in the evaluation (Figure 139A). So that you can steer clear of exclusion of activated/proliferating B cells, the FSC gate really should be not also restrictive. Exclusion of dead cells by way of application of live/dead discrimination reagents is strongly advisable [1], this measure is of important value particularly when smaller sized subpopulations are included in the analyses. The spleen includes MZ B cells which might be unique to this organ. The immature B cells stages T1, T2 and T3 are also selectively located inside the spleen. In contrast, lymph nodes and Peyer’s patches contain neither MZ nor immature B cells, but harbor mainly follicular B cells. In spleen and also other secondary lymphoid tissues, all B cells are CD19pos and B220pos (of note, not all plasma cells express these two markers, see Chapter VI Section three.1 Murine Absecreting plasmablasts and plasma cells). For that reason, CD19 or B220 might be used as alternative markers for the identification of B lineage cells in these tissues. In spleen, staining for B220 (or CD19), CD21, CD23 and IgM permits identification of follicular B cells and MZ B cells [1132, 1133]. We also recommend to stain furthermore for IgD. Using this marker mixture, follicular B cells are identified by their B220pos/CD21intmed/ CD23high phenotype, MZ B cells are B220pos/CD21high/CD23low/neg (Fig. 139B). Though their characteristic B220/CD21/CD23 expression profile is sufficient to recognize follicular and MZ B cells, their identity is usually additional proofed by their distinct IgDpos/IgMintmed and IgDlow/neg/IgMhigh phenotype, respectively (Fig. 139C). After further gating B220pos cells on IgM vs CD21 and CD23, this marker combination also allows to determine T1 and T2 cells [1134]. All secondary lymphoid organs can contain GCs where B cells can create Abs of enhanced affinity, just after correct stimulation within the context of a T-dependent immunization. GCs are transient structures present soon after immunization with T-dependent (protein) antigens whichAuthor Manuscript Author Manuscript Author Manuscript Author ManuscriptEur J NOX4 Inhibitor supplier Immunol. Author manuscript; α adrenergic receptor Antagonist manufacturer available in PMC 2020 July ten.Cossarizza et al.Pageare absent in steady state. Flow cytometric analysis of GC B cells is described in section Chapter VI Section two.2 Murine Germinal Center B cells. Ultimately, the GC reaction provides rise to plasma cells and memory B cells. Plasma cells are described in detail in Section three of this chapter (Murine Ab-secreting plasmablasts and plasma cells. Memory B cells are identified in spleen and in the peripheral blood. The murine B cell memory compartment appears in several subsets and exhibits a very heterogeneous phenotype [1135]. Memory B cells specific for one particular distinct antigen is usually identified by staining with fluorescent-labeled antigen. Nonetheless, due to the low frequencies of those cells and unspecific binding to other B cells, this method is challenging and needs careful controls [1136, 1137]. Usage of adoptive transfer of B cells from BCR trans.