B cells

1 Introduction

B cells are white blood cells of the lymphocyte subtype (PMID: 18897986). They are key players in the adaptive immune response exerting a 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 the pathogenesis of various autoimmune diseases. B cell malignancies are often characterized by loss or gain of expression of surface markers compared to corresponding normal B cells.

2 B Cells from lymphoid tissues

B cell development takes place in bone marrow. After expression of a functional, non-autoreactive B cell receptor (BCR), naive B cells leave the bone marrow and circulate through the body via the blood stream. Upon activation, B cells either develop into plasma cells or migrate into secondary lymphoid organs like tonsils, spleen, or Peyer’s patches for further differentiation (PMID: 18725575, 17582343). All mature B cells express the Pan B cell markers CD19, CD20, and CD22 (PMID: 1373518, 26478008, 21151033).

2.1 Cell subsets, frequencies, and marker expression

At a glance: B cell subsets in lymphoid tissue

Cell subsetMarkersFunction
B-1 B cellsCD138, CD43, CD5• Involved in innate-like immune response
• Produce natural antibodies
B-2 B cellssee table below• Give rise to marginal zone and follicular B cells 
• Progenitors of various developmental B cell subsets

At a glance: B-2 B cell subsets in lymphoid tissue

Cell subsetMarkersFunction
Follicular B cellsIgMlow, CD45Rhigh, CD38, CD21, CD23, CD22, CD19, Pax5Shuttling between bone marrow and secondary lymphoid organs
Marginal zone B cellsIgMhigh, IgDlow, CD1d, CD9, CD21, CD22, CD35, CD45R, CD23, Pax5, EBF, E2A, Oct2Secondary lymphoid organs
Transitional/immature B cellsB220, CD93, CD24high, IgM, BR3, TACI, Pax5, EBF, E2A, Oct2Migration from bone marrow to secondary lymphoid organs
Germinal center B cellsCD45R, GL7, CD95, PNA, BR3, IgD, IgM, BCL6, EBFSecondary lymphoid organs
Plasma cellsIgD, CD45Rlow, CD138high, TACI and/or BCMA, CD126, CD184, CD320, BLIMP1, IRF4, XBP1Long-lived plasma cells in bone marrow. Short-lived plasma cells in secondary lymphoid organs
Memory B cellsCD45R, CD80, CD73, CD273, CD38, CD84, CD86, Pax5, PBF1, SPI-BCirculating in both bone marrow and lymphoid tissue

Note: Frequencies of distinct subsets vary depending on mouse strain, age, and several other factors. Therefore, an average number cannot be provided (PMID: 18544662).

There are three principal classes of B lymphocytes: B-1 B cells, and B-2–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 (B reg) cells. Mouse B cells are additionally classified into transitional/immature, naive B cells, memory B cells, and plasma cells. Further subsets of memory B cells and plasma cells are identified based on their Ig isotype expression (IgM, IgD, IgG, IgA).

B-1 B cells develop from the fetal liver and disseminate into the periphery. They are principally found in the pleural and peritoneal cavities. Based on the surface expression of CD138, CD43, and CD5, two distinct subsets can be identified: B-1a and B-1b. B-1a B cells are the most abundant and express CD5, whereas B-1b B cells lack CD5 expression. B-1 cells 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 the 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 lymphocyte subsets, i.e., naive, activated, germinal center, memory, and plasma cells (PMID: 22224772).

B cells in lymphoid organs reside in structures called follicles. In response to antigen encounter within the follicle, naive B cells initiate the formation of germinal centers. 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 germinal centers, 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 germinal center 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.

2.2 Miltenyi Biotec application protocols for B cells from lymphoid tissue

Miltenyi Biotec has created dedicated application protocols for working with mouse B cells.

2.3 Sample preparation of lymphoid tissues

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. 

2.4 Magnetic separation of B cells from lymphoid tissue

Miltenyi Biotec has developed numerous products for the magnetic separation of the various B cell types and subsets that can be found in mouse lymphoid tissue. For details on MACS® Cell Separation Technology, see the MACS Handbook chapter Magnetic cell separation .

2.4.1 Isolation of Pan B cells

At a glance: Kits and reagents for the separation of Pan B cells from lymphoid tissue

Starting materialIsolation strategyCommentsAutomation  Product
Single-cell suspensions from bone marrow, lymph node, spleen, as well as lung, lamina propria, pleural and peritoneal cavity tissuesPositive selection of target cells CD45R is expressed throughout B cell development. Also expressed by plasmacytoid DCsYes*CD45R (B220) MicroBeads, mouse
Single-cell suspensions from  lymph node, spleen, and bone marrowPositive selection of target cells Yes*CD19 MicroBeads, mouse
Single-cell suspensions from lymph node, spleen, and bone marrow, as well as bloodPositive selection of target cells Yes*CD22 MicroBeads, mouse
Single-cell suspensions from lymph node and spleenDepletion of non-target cellsSuited to isolate B cells from mouse models of human disease, e.g., B-CLL mouse modelsYes*Pan B Cell Isolation Kit II, mouse
*Automation options range from fully automated benchtop solutions such as the autoMACS® Pro Separator to high-throughput platforms such as the MultiMACS™ Cell24 Separator Plus or MultiMACS X

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 areas of research.

Depending on experiment objectives, all B cells can be isolated from single-cell suspensions of lymphoid tissue by positive selection using CD45R (B220), CD19, or 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. Because the depletion cocktail of this kit does not include antibodies against CD43 or CD11b, which might be expressed on malignant target cells, the kit is optimal for use with different mouse models of human diseases.

Pan B Cell Isolation Kit II, mouse
Before separation
After separation

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-APC and CD4-FITC and analyzed by flow cytometry using a MACSQuant Analyzer. Cell debris and dead cells were excluded from the analysis based on scatter signals and propidium iodide fluorescence.

2.4.2 Isolation of B cell subsets

At a glance: Kits and reagents for the separation of various B cell subsets from lymphoid tissue

B cell subtypeStarting 
material
Isolation 
strategy
CommentsAutomation  Product
B-1a cellsSingle-cell suspensions from lymphoid organs, non-lymphoid tissue, bloodPositive selection of target cells  Yes*CD5 (Ly-1) MicroBeads, mouse
B-1a cellsSingle-cell suspensions from mouse body cavities or spleenDepletion of non-target cells followed by positive selection of target cellsBased on expression of CD5Yes*B-1a Cell Isolation Kit, mouse
Regulatory B cellsSingle-cell suspensions from lymphoid organs    Depletion of non-target cells followed by stimulation of enriched B cells and isolation of IL-10–secreting cellsIsolation of IL-10–producing cells is done with Cytokine Secretion Assay technologyYes*Regulatory B Cell Isolation Kit, mouse
B-2 cell subsets
Mature (resting) B cellsSingle-cell suspensions from lymphoid tissues    Positive selection of target cellsBased on expression of CD23 antigenYes*CD23 MicroBeads, mouse
Mature (resting) B cellsSingle-cell suspensions from lymphoid tissues    Depletion of non-target cellsTwo-step labeling and depletion of CD43-expressing B cells and non-B cellsYes*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 cellsYes*MZ and FO B Cell Isolation Kit, mouse
Mature (resting) B cellsSingle-cell suspensions from lymphoid tissueDepletion of non-target cellsImmature and mature resting B cells do not express CD43Yes*CD43 (Ly-48) MicroBeads, mouse
Memory B cellsSingle-cell suspensions from spleen or lymph nodesDepletion of non-target cells followed by positive selection of target cellsBased on expression of CD27 on most memory B cellsYes*Memory B Cell Isolation Kit, mouse
Memory B cellsSingle-cell suspensions from lymphoid tissues    Positive selection of target cellsIsolates B cells expressing IgG1Yes*Anti-Mouse IgG1 MicroBeads
Memory B cellsSingle-cell suspensions from lymphoid tissues    Positive selection of target cellsIsolates B cells expressing IgG2a or IgG2bYes*Anti-Mouse IgG2a+b MicroBeads
Transitional B cellsSingle-cell suspensions of bone marrow from adult mice and fetal liver from unborn micePositive selection of target cellsIsolates CD93-expressing B cell progenitors that have migrated to lymphoid tissueYes*CD93 MicroBeads, mouse
Plasma cellsSingle-cell suspensions from spleen or lymph nodesDepletion of non-target cells followed by positive selection of target cellsCD138 is re-expressed in plasmablasts and plasma cells upon B cell differentiationYes*CD138+ Plasma Cell Isolation Kit, mouse
Plasma cellsSingle-cell suspensions from spleenPositive selection of target cells Yes*CD138 MicroBeads, mouse
Germinal center B cellsSingle-cell suspensions from lymphoid tissues after immunizationPositive selection of target cellsBased on expression of ligands for peanut agglutinin (PNA)Yes*Germinal Center B Cell (PNA) MicroBead Kit, mouse
*Automation options range from fully automated benchtop solutions such as the autoMACS® Pro Separator to high-throughput platforms such as the MultiMACS™ Cell24 Separator Plus or MultiMACS X

The B Cell Isolation Kit, mouse has an updated, rapid protocol that enables isolation of 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.

View details

CD45R+ B cells are isolated at a high recovery rate. Untouched B cells were isolated from a single-cell suspension from mouse spleen using the B Cell Isolation Kit, a MidiMACS Separator, and an LS Column.

The CD138+ Plasma Cell Isolation Kit, mouse leverages the correlation between expression of CD138 on cells of the B cell lineage and their developmental stage, location, and adhesion. CD138 is expressed on pre-B and immature B lymphocytes in bone marrow, whereas circulating and peripheral B cells do not express this antigen. Upon differentiation of B cells, plasmablasts and plasma cells re-express CD138.

View details

CD138+ plasma cells are efficiently isolated in a two-step process. Cells were isolated from a single-cell suspension from mouse spleen using the CD138+ Plasma Cell Isolation Kit, an LD and two MS Columns, a MidiMACS™ Separator, and a MiniMACS™ Separator. The cells were fluorescently stained with CD45R (B220)-APC, CD19-FITC, and CD138-PE. Cell debris and dead cells were excluded from the analysis based on scatter signals and propidium iodide fluorescence.

Characterization of B cells in the germinal center can support the development of improved vaccine strategies and result in a better understanding of B cell diseases. B cells within the germinal center express ligands for peanut agglutinin (PNA), allowing for B cell identification and isolation. PNA is a plant lectin derived from the fruits of Arachis hypogae, which specifically binds to terminal non-reducing galactose residues on the cell membrane. This affinity for PNA is leveraged by the new Germinal Center B Cell (PNA) MicroBead Kit, mouse to separate germinal center B cells after primary immunization with T-dependent antigens.

View details

 

Isolation of germinal center B cells from SRBC-immunized mouse spleen. 
Cells were separated using the Germinal Center B Cell (PNA) MicroBead Kit, an MS Column, and a MiniMACS Separator. Cells were fluorescently stained with CD19-PE-Vio770, CD38-APC, and CD95-PE and analyzed by flow cytometry using a MACSQuant Analyzer. Cell debris and dead cells were excluded from the analysis based on scatter signals and propidium iodide fluorescence.
 


 

 

2.4.3 Conjugated MicroBeads for the separation of B cell subsets

For the isolation of B cell subsets that are not defined by a single antigen and thus are not amenable to a single, specific Miltenyi Biotec solution, we offer a range of MicroBeads for direct or indirect magnetic labeling that can be combined to separate the subset of choice. MicroBeads for indirect magnetic labeling, namely Anti-FITC and Anti-PE MicroBeads, have been used to separate CD23+ follicular B cells and CD21+CD23 MZ B cells from mouse spleen (PMID: 16880262).

2.5 Characterization of B cells by flow cytometry

Miltenyi Biotec offers a broad portfolio of products for the routine cell surface and intracellular staining of B cell–specific markers to examine B cell populations and subsets by flow cytometry. The vast portfolio of recombinant REAfinity™ Antibodies enables comprehensive B cell analysis.

2.5.1 Flow cytometry panels

The antibody panel builder facilitates planning and ordering of customized panels.

Related PDFs:
Related documents:
Related products:
2.5.2 Analysis of cytokines

At a glance: Kits and reagents for the analysis of cytokine secretion from B cells

UseCommentsProduct
Detection of IL-10–secreting cells Mouse IL-10 Secretion Assay – Detection Kit (PE)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)
Measuring the concentration of ten selected cytokines in one sampleOptimized for automated measurementMACSPlex Cytokine 10 Kit, mouse
Measuring the concentration of up to seven cytokines in one sampleContains supplementary components to perform a cytokine assay; to be combined with a MACSPlex Cytokine Standard and MACSPlex Cytokine Reagents KitMACSPlex Cytokine Basic Kit, human and mouse

MACS Cytokine Secretion Assays enable the easy enrichment, analysis, and enumeration of viable cytokine-secreting cells. These unique tools allow the detection of cytokine secretion at the single-cell level and multiplexed phenotyping. As an example, the Mouse IL-10 Secretion Assay was instrumental in showing that in neonatal spleen cells activated by CpG oligodeoxynucleotides, CD45R+ and CD19+ B cells and not CD3+ T or CD11b+ myeloid cells were primarily responsible for the production of IL-10 (PMID: 15845451).

View details

Principle of the Cytokine Secretion Assay – Cell Enrichment and Detection Kits.

2.6 Cell culture and expansion of B cells

Miltenyi Biotec offers a broad portfolio of over 150 cytokines and growth factors as well as related proteins to culture or stimulate B cells, and to investigate their function. For example, Miltenyi Biotec's high-quality cytokines and CpG oligodeoxynucleotides are perfectly suited for the activation and expansion of mouse B cells for further studies.

MACS Handbook:

Cell culture

3 B cells from bone marrow

B cell development in bone marrow is a tightly regulated process, with stepwise recombination of V, (D) and J gene segments coding for the variable (V) region of the immunoglobulin (Ig) heavy and light chains. Throughout adulthood, pre-B and immature B lymphocytes can be found in bone marrow. For additional information about B cells in bone marrow, see the chapter on B cells from lymphoid tissues.

3.1 Sample preparation of bone marrow

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.

3.2 Magnetic separation of B cells from bone marrow

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.
 

3.2.1 Isolation of B cells from bone marrow

In mice, CD138 is expressed on pre-B and immature B lymphocytes in the bone marrow. CD138 expression is lost when B cells emigrate into the periphery, and is absent on circulating and peripheral B cells. Only upon differentiation into plasmablasts and plasma cells, B cells re-express CD138 (PMID: 2519615). Thus, the CD138+ Plasma Cell Isolation Kit, mouse is optimal for the isolation of CD138+CD45R(B220)low/–CD19low/– cells in bone marrow.