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B cells are white blood cell of the lymphocyte subtype (PMID: 18897986). They are key players in the adaptive immune response for their critical role in generating humoral immunity. Additionally, they contribute directly to cellular immunity as bona fide effectors, secreting inflammatory cytokines, serving as professional antigen-presenting cells (APCs), and modulating immune responses as regulatory B cells (PMID: 25324123). They also maintain the architecture of secondary lymphoid organs and promote the generation of tertiary lymphoid tissues at sites of chronic inflammation (PMID: 22224772).B cells have been implicated in several malignancies and pathogenesis of various autoimmune diseases. B cell malignancies are often characterized by lost or gained expression of surface markers compared to corresponding normal B cells.
|B-1 B cells||CD138, CD43, CD5|
|B-2 B cells|
|Subsets of B-2 B cells|
|Follicular B cells||IgMlow, CD45Rhigh, CD38, CD21, CD23, CD22, CD19, Pax5||Shuttling between bone marrow and secondary lymphoid organs|
|Marginal zone B cells||IgMhigh, IgDlow, CD1d, CD9, CD21, CD22, CD35, CD45R, CD23, Pax5, EBF, E2A, Oct2||Secondary lymphoid organs|
|Transitional/immature B cells||B220, CD93, CD24high, IgM, BR3, TACI, Pax5, EBF, E2A, Oct2||Migration from bone marrow to secondary lymphoid organs|
|Germinal center B cells||CD45R, GL7, CD95, PNA, BR3, IgD–, IgM–, BCL6, EBF||Secondary lymphoid organs|
|Plasma cells||IgD–, CD45Rlow, CD138high, TACI and/or BCMA, CD126, CD184, CD320, BLIMP1, IRF4, XBP1||Long-lived plasma cells in bone marrow. Short-lived plasma cells in secondary lymphoid organs|
|Memory B cells||CD45R, CD80, CD73, CD273, CD38, CD84, CD86, Pax5, PBF1, SPI-B||Circulating in both bone marrow and lymphoid tissue|
Note: Frequencies of distinct subsets varies according to mouse strain, age, and several other factors, thus an average number cannot be provided (PMID: 18544662).
There are 3 principal classes of B lymphocytes: B1 B cells, and B2-derived marginal zone (MZ) or follicular (FO) B cells (PMID: 21151033). A small percentage of these B cells produce IL-10 and are referred to as regulatory B cells (B reg). Mouse B cells are additionally classified as transitional/immature, naïve B cells, memory B cells, and plasma cells. Further subsets of memory B cells and plasma cells are identified based on their Ig isotypes expression (IgM, IgD, IgG, IgA).
B-1 B cells develop from the fetal liver and disseminate to the periphery. They are principally found in the pleural and peritoneal cavities. Based on the surface expression of CD138, CD43 and CD5, 2 distinct subsets are recognized: B-1a and B-1b. B-1a B cells are the most abundant and express CD5. B1 cells lack CD5 expression. They are involved in innate-like immune responses and are the main producers of natural antibodies. Interestingly, they express specific TLRs and thus, can mount a response in absence of (BCR) triggering.
B-2 B cells develop from bone marrow, migrate to the periphery as immature B cells, and mature once they reach the spleen or other secondary lymphoid organs. They can give rise to both marginal zone (MZ) or follicular B cells (FO). The latter are considered the conventional B cell subset and are progenitors for the developmental B lymphocytes subsets naïve, activated, GC, memory, and plasma cells (PMID: 22224772).B cells in lymphoid organs reside in structures called follicles. In response to antigen encounter within the follicle, naïve B cells initiate the formation of germinal centers (GC). These transient structures are critical for the development of the adaptive humoral response, and are the location where B cells differentiate and proliferate to generate long-lasting immunological memory. Within GCs, B cells undergo IgV somatic hypermutation, affinity maturation, and Ig class switching, leading to the development of switched memory B cells and plasma cells. Elucidating the dynamics and mechanics of the GC reaction is a major research subject in adaptive immunity, immunodeficiency, and B cell diseases, and of critical importance for the development of improved vaccination strategies.
Lymphoid tissues must be dissociated into a single-cell suspension for many downstream applications, including isolation of cell subpopulations, cell culture, or flow cytometry analysis. Dissociation can be accomplished fully automatically using a gentleMACS™ Dissociator and specific Tissue Dissociation Kits (e.g., Spleen Dissociation Kit, mouse). A special protocol for the preparation of single-cell suspensions from mouse spleen without enzymatic treatment can be downloaded from the Related Resources panel to the right. Alternatively, tissues can be dissociated using a manual procedure.For details about sample preparation of lymphoid tissues and bone marrow, see the corresponding chapters.
|Starting material||Isolation strategy||Comments||Automation||Product|
|Single-cell suspension of bone marrow, lymph node, spleen, as well as lung, lamina propria, pleural and peritoneal cavity tissues||Positive selection of target cells||Marker expressed throughout B cell development. Also expressed by plasmacytoid DCs||Yes|
|Single-cell suspension of lymph node, spleen and bone marrow||Positive selection of target cells||Yes||CD19 MicroBeads, mouse|
|Single-cell suspension of lymph node, spleen and bone marrow, as well as blood||Positive selection of target cells||Yes||CD22 MicroBeads, mouse|
|Single-cell suspension from lymph node and spleen||Depletion of non-target cells||Suited to isolate B cells from mouse models of human disease, e.g., B-CLL mouse models||Yes||Pan B Cell Isolation Kit II, mouse|
B cell biology and mechanisms of action are still actively researched, and the analysis of whole B cell compartments enables elucidating signal requirements for B cell activation, induction of proliferation, and differentiation. Signal transduction, immunoglobulin class switching, and somatic hypermutation in B cells are other active areas of research.Depending on experiment objectives, all B cells can be isolated from single-cell suspensions of lymphoid tissue by positive selection using the CD45R (B220), the CD19, or the CD22 Microbeads, mouse. Alternatively, the Pan B Cell Isolation Kit II, mouse offers a fast and efficient method to separate untouched B-1 and B-2 B cell subsets, and because the kit depletion cocktail does not include CD43 or CD11b, which might be expressed on malignant target cells, the kit is optimal for use with different mouse models of human diseases.
Isolation of untouched B cells. Pan B cells were isolated from mouse spleen using the Pan B Cell Isolation Kit, an LD Column, and a MidiMACS™ Separator. The cells were fluorescently stained with CD19-PE and CD4-FITC and analyzed by flow cytometry using the MACSQuant Analyzer. Cell debris and dead cells were excluded from the analysis based on scatter signals and propidium iodide fluorescence.
|B cell subtype||Starting |
|B-1a cells||Single-cell suspension of lymphoid organs, non-lymphoid tissue, blood||Positive selection of target cells||Yes|
|B-1a cells||Single-cell suspension of mouse body cavities or spleen||Depletion of non-target cells followed by positive selection of target cells||Based on expression of CD5||Yes||B-1a Cell Isolation Kit, mouse|
|Regulatory B cells||Single-cell suspension of lymphoid organs||Depletion of non-target cells followed by stimulation of enriched B cells and isolation of IL-10 secreting cells||Isolation of IL-10–producing cells is done with Cytokine Secretion Assay technology||Yes||Regulatory B Cell Isolation Kit, mouse|
|B-2 cell subsets|
|Mature (resting) B cells||Single-cell suspension of lymphoid tissues||Positive selection of target cells||Based on expression of CD23 antigen||Yes||CD23 MicroBeads, mouse|
|Mature (resting) B cells||Single-cell suspension of lymphoid tissues||Depletion of non-target cells||Two-step labeling and depletion of CD43-expressing B cells and non-B cells||Yes||B Cell Isolation Kit, mouse|
|Mature (resting) B cells||Depletion of non-target cells followed by positive selection of FO B cells.||Delivers two fractions: FO B cells and MZ B cells||Yes||MZ and FO B Cell Isolation Kit, mouse|
|Mature (resting) B cells||Single-cell suspension of lymphoid tissue||Depletion of non-target cells||Immature and mature resting B cells do not express CD43||Yes||CD43 (Ly-48) MicroBeads, mouse|
|Memory B cells||Single-cell suspension of spleen or lymph nodes||Depletion of non-target cells followed by positive selection of target cells||Based on expression of CD27 on most memory B cells||Yes||Memory B Cell Isolation Kit, mouse|
|Memory B cells||Single-cell suspension of lymphoid tissues||Positive selection of target cells||Isolates B cells expressing IgG1||Anti-Mouse IgG1 MicroBeads|
|Memory B cells||Single-cell suspension of lymphoid tissues||Positive selection of target cells||Isolates B cells expressing IgG2a or IgG2b||Anti-Mouse IgG2a+b MicroBeads|
|Transitional B cells||Single-cell suspension of bone marrow from adult mice and fetal liver from unborn mice||Positive selection of target cells||Isolates CD93-expressing B cell progenitors that have migrated to lymphoid tissue||Yes||CD93 MicroBeads, mouse|
|Plasma cells||Single-cell suspension of spleen or lymph nodes||Depletion of non-target cells flowed by positive selection of target cells||Plasmablasts and plasma cells recover CD138 expression||Yes||CD138+ Plasma Cell Isolation Kit, mouse|
|Plasma cells||Single-cell suspension of spleen||Positive selection of target cells||CD138 MicroBeads, mouse|
|Germinal center B cells||Single-cell suspension of lymphoid tissues after immunization||Positive selection of target cells||Based on expression of ligands for peanut agglutinin (PNA)||Yes||Germinal Center B Cell (PNA) MicroBead Kit, mouse|
The B Cell Isolation Kit, mouse has an updated, rapid protocol that enables isolating untouched resting B cells using cocktails designed to deplete activated B cells, plasma cells, CD5+ B-1a cells, and non-B cells. The isolated B cells exhibit high cell viability and recovery.
|B cell subset||Markers||Suggested antibody|
CD19-VioBlue, mouse (clone: REA749)
CD45R (B220)-PE-Vio615, mouse (clone: REA755)CD5-APC, mouse (clone: REA421)
CD45R (B220)-PE-Vio615, mouse (clone: REA755)CD23-PE, mouse (clone: B3B4)
|Detection of IL-10-secreting cells||Mouse IL-10 Secretion Assay – Detection Kit (APC)|
|Detection and enrichment of IL-10 secreting cells||Mouse IL-10 Secretion Assay – Cell Enrichment and Detection Kit (PE)|
|Determine concentration of 10 selected cytokines in one sample||Optimized for automated measurement||MACSPlex Cytokine 10 Kit, mouse|
|Determine concentration of up to 7 cytokines in one sample||Contains supplementary components to perform a cytokine assay; to be combined with a MACSPlex Cytokine Standard and MACSPlex Cytokine Reagents Kit||MACSPlex Cytokine Basic Kit, human and mouse|
Mononuclear cells are typically isolated from bone marrow before subsequent separation of B cells.For details about sample preparation of lymphoid tissues and bone marrow, see the corresponding MACS Handbook chapters..
Miltenyi Biotec has developed numerous products for the magnetic separation of the various B cell types and subsets. For details on MACS® Cell Separation Technology, see the MACS Handbook chapter Magnetic Cell Separation .