MACS Handbook

B cells

1 Introduction

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.

2 B Cells from lymphoid tissues

B cell development takes place in bone marrow, but after expression of a functional,non-autoreactive B cell receptor (BCR), naïve 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 cells
  • Give rise to marginal zone and follicular B cells
  • Progenitors of various developmental B cell subsets
Subsets of B-2 B cells
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 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.

2.2 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. 

2.3 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 .
2.3.1 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 suspension of bone marrow, lymph node, spleen, as well as lung, lamina propria, pleural and peritoneal cavity tissuesPositive selection of target cells Marker expressed throughout B cell development. Also expressed by plasmacytoid DCsYes*

CD45R (B220) MicroBeads, mouse

Single-cell suspension of lymph node, spleen and bone marrowPositive selection of target cellsYes*CD19 MicroBeads, mouse
Single-cell suspension of lymph node, spleen and bone marrow, as well as bloodPositive selection of target cellsYes*CD22 MicroBeads, mouse
Single-cell suspension 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 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.
Pan B Cell Isolation Kit II, mouse

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.

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2.3.2 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 suspension of lymphoid organs, non-lymphoid tissue, bloodPositive selection of target cells Yes*

CD5 (Ly-1) MicroBeads, mouse

B-1a cellsSingle-cell suspension of 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 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 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 cells Single-cell suspension of lymphoid tissues    Positive selection of target cellsBased on expression of CD23 antigenYes*CD23 MicroBeads, mouse
Mature (resting) B cells Single-cell suspension of 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 cellsDepletion 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 suspension of lymphoid tissueDepletion of non-target cellsImmature and mature resting B cells do not express CD43Yes*CD43 (Ly-48) MicroBeads, mouse
Memory B cellsSingle-cell suspension of 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 cells Single-cell suspension of lymphoid tissues    Positive selection of target cellsIsolates B cells expressing IgG1Yes*Anti-Mouse IgG1 MicroBeads
Memory B cells Single-cell suspension of lymphoid tissues    Positive selection of target cellsIsolates B cells expressing IgG2a or IgG2bYes*Anti-Mouse IgG2a+b MicroBeads
Transitional B cellsSingle-cell suspension 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 suspension of spleen or lymph nodesDepletion of non-target cells flowed by positive selection of target cellsPlasmablasts and plasma cells recover CD138 expressionYes*CD138+ Plasma Cell Isolation Kit, mouse
Plasma cellsSingle-cell suspension of spleenPositive selection of target cellsYes*CD138 MicroBeads, mouse
Germinal center B cellsSingle-cell suspension of 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 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.

View details

High recovery of isolated BC45R+ B cells. Untouched B cells were isolated from a mouse spleen single-cell suspension 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. First expressed on pre-B and immature B lymphocytes in bone marrow, CD138 expression is lost in circulating and peripheral B cells. Upon differentiation of B cells into plasmablasts and plasma cells, expression of CD138 is recovered.
View details

CD138+ Plasma cells efficiently isolated in a two-step process. Cells were isolated from a mouse spleen single-cell suspension using the CD138+ Plasma Cell Isolation Kit, an LD and two MS Columns, a MidiMACS 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.

Examining and characterizing B cells in GC can support improved vaccine strategies and a better understanding of B cell diseases. B cells within GC express ligands for peanut agglutinin (PNA), allowing for their identification and isolation. PNA is a plant lectin protein 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 our new Germinal Center B Cell PNA MicroBead Kit, mouse, to separate GC B cells following primary immunization with T-dependent antigens.
View details

Isolation of germinal center B cells from mouse SRBC-immunized spleen. 
Cells were separated using the Germinal Center B Cell (PNA) MicroBead Kit, an MS Column, and a MiniMACS Separator. Cells are fluorescently stained with CD19-PE-Vio® 770, CD38-APC, and CD95-PE and analyzed by flow cytometer using the MACSQuant Analyzer. Cell debris and dead cells were excluded from the analysis based on scatter signals and propidium iodide fluorescence.


2.3.3 Conjugated MicroBeads for the separation of B cell subsets
To isolate B cell subsets that are not defined by a single antigen or covered by a specific Miltenyi Biotec solution, we offer a range of directly conjugated MicroBeads and indirect MicroBeads 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).

2.4 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 that can be used to examine B cell populations and subsets by flow cytometry. Our innovativeREAfinity™ antibodies  are ideal for the analysis of B cells with the MACSQuant® Analyzer.
2.4.1 Flow cytometry panels

At a glance: Markers and suggested panels for the characterization of B cells by flow cytometry

Use MACS Flow Cytometry – multicolor Panel Builder to plan and order customized panels.
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2.4.2 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 cellsMouse IL-10 Secretion Assay – Cell Enrichment and Detection Kit (PE)
Determine concentration of 10 selected cytokines in one sampleOptimized for automated measurementMACSPlex Cytokine 10 Kit, mouse
Determine concentration of up to 7 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
MACSCytokine Assays (CSA) enable the easy enrichment, analysis, and enumeration of viable cytokine-secreting cells. These unique tools, allow the detection of cytokine secretion at single-cells 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.

2.5 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 and stimulate B cells, and to investigate their function. For example, our 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 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. This 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, do B cells express CD138 again (PMID: 2519615). Thus, our CD138+ Plasma Cell Isolation Kit, mouse is optimal for the isolation of CD138+CD45R(B220)low/–CD19low/– in bone marrow.