MACS Handbook

B cells

1 Introduction

B cells, also known as B lymphocytes, belong to the white blood cells. They secrete antibodies and are a part of the adaptive immune system. B cells also play a role as professional antigen-presenting cells (APCs) and secrete cytokines.

2 B cells in peripheral blood

B cells circulate through the body via the peripheral blood. Among all peripheral blood lymphocytes, B cells account for 2-10% of all lymphocytes. All mature B cells in blood express the Pan B cell marker CD19, CD20 and CD22. (PMID: 16227086, 7524522)

Peripheral blood B cells can be classified into transitional/immature, naive and memory B cells, and plasma cells. Additionally, different subsets of memory B cells and plasma cells can be identified based on their expression of Ig isotypes (IgM, IgD, IgG, IgA).

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2.1 Cell subsets, frequencies, and marker expression

Peripheral blood includes several B cell subsets (PMID : 18434123, 22566870):
2.1.1 Naive B cells
If a B cell succeeds in producing a functional, non-autoreactive B-cell receptor (BCR), it differentiates into a mature, naïve B cell. These cells express the BCR as IgM and IgD molecules, recirculate through the body, and account for roughly 60–70% of B cells in adults. Naïve B cells are CD27, a receptor of the TNF family that plays a role in B cell activation and subsequent immunoglobulin synthesis.
2.1.2 Activated B cells
B cells activation requires two signals to obtain the clustering of B cell receptors and their associated signaling molecules. This first is initialized by the binding of a specific antigen to the BCR. The second signal is costimulatory and can be T cell-dependent or T cell-independent. For T cell-dependent costimulation, the TCR of T helper cells recognize the same antigen as the respective B cells, which the B cell has processed and presented on its surface in a Ag:MHCII complex beforehand. This in turn activates the T cell, which then provides a second activation signal for the B cell, like the expression of CD40L on the T helper cell surface. CD40L can bind to CD40 on B cells enabling increased clustering and multimerization of the receptors (PMID: 27800605), which triggers a signaling cascade that increases proliferation, possibly induces class switch, and drives differentiation to plasma or memory cells. T cell independent costimulation normally involves antigens which exhibit a structure that can already multimerize the BCR without the need of T cell support. These antigens display highly repetitive epitope motifs, like bacterial cell wall components (carbohydrates and lipopolysaccharides). Activated B cells display an increase in their expression of CD86, CD80, and MHCII or CD62L, which can be detected by flow cytometry.
2.1.3 Memory B cells

Memory B cells are generated in germinal center (GC) reactions in the course of T cell-dependent immune responses and are distinguished from naive B cells by an increased lifespan, faster and stronger response to stimulation, and expression of somatically mutated and affinity-matured immunoglobulin (Ig) genes. Approximately 15–30% of human B cells in adults are memory B cells, and several subsets have been identified. Besides IgG+ and IgA+ memory B cells, around 25% of peripheral blood memory B cells express IgM with or without IgD. Additional subpopulations have been described. These subsets share features of memory B cells, but likely fulfill distinct functions. 
In humans, the tumor necrosis factor receptor superfamily member CD27 has become an important marker to distinguish naïve from mutated IgM+ memory B cells (PMID: 22566870). NB GC B cells and post-GC PCs are also CD27+ but can be excluded from analysis by gating out CD38++ cells.


CD27 expression on selected B cell subsets (PMID: 27499139)

 FeatureNaive B cellsGC B cellsIgM+IgD+CD27+ cellsIgM-onlyIgG+ and IgA+
Frequency in peripheral blood50%NA15%5%25%
Ig isotype expressionIgM, IgDIgM or IgDIgM, IgDIgMIgG or IgA
CD27 expression-++++
Life spanWeeksShort-livedLong-livedLong-livedLong-lived

IgG memory B cells

Approximately 15–20% of peripheral blood (PB) B cells in adults are IgG+ B cells, expressing mainly IgG1, IgG2 or IgG3, and rarely IgG4. Proportions of these cells among memory B cells vary considerably among individuals, likely because of different infection histories. Switching to specific IgG subclasses is influenced by the type of antigen and various immunoregulatory factors

Approximately 20–25% of IgG memory B cells lack CD27 expression (PMID:(PMID: 22566870). This subpopulation increases in the elderly suggesting these may be aged or exhausted memory B cells. IgG+CD27− B cells are considerably less mutated than their CD27+ counterparts, and are more frequently IgG3+ and less frequently IgG2+. Both types of memory B cells often derive from common GC B-cell clones.

In mice and humans, IgG memory B cells have the propensity to differentiate into PCs upon reactivation, and mostly not to reenter GC reactions

IgA memory B cells

IgA memory B cells account for approximately 10% of B cells in peripheral blood and mostly express CD27. They are preferentially generated in immune responses in the intestine and other mucosa-associated lymphatic tissues, like Peyer’s patches or mesenteric lymph. 

IgE memory B cells

IgE-expressing memory B cells are hardly detectable in the peripheral blood of healthy humans. They are important in immunologic reactions against parasites like helminths and protozoa, and their interaction with mast cells and eosinophils generates allergic reactions.

IgM-only and IgM+IgD+ memory B cells

Two distinct subpopulations of human IgM-expressing PB B cells with somatically mutated IgV genes exist, IgM-only B cells (IgM+IgDlow/−) and IgM+IgD+ B cells, both expressing CD27 and representing roughly 5% and 15% of B cells, respectively, in PB and secondary lymphoid organs, hence representing a major fraction of the B-cell pool.. In course of an adaptive immune response all mature B cells are IgM+ or IgD+ at first and undergo class switch to other Isotypes classes only later depending on the surrounding influences, mediated by cytokines. IgM+ cells are mostly involved in immune response which involve and activate the complement system.

2.1.4 Plasma cells
Human plasma cells are a heterogeneous cell population comprising several subsets, from short-lived and proliferative plasmablasts in lymphoid tissues, to transitional plasma cells in peripheral blood, and long-lived, non-dividing plasma cells in bone marrow (PMID: 28633925). Plasma cells of all differentiation stages are identified by high-level expression of CD38 (PMID: 28633925). Other surface markers are differentially regulated, depending on stage of differentiation and spatial localization. Plasma cells in blood lack expression of the typical B-cell marker CD22 and express lower levels of CD19 and CD20 than mature B cells. They are further characterized as being CD27++CD31+CD44+CD45+CD56CD62L+CD86+ and HLA-DR+. A subset of CD38+ blood plasma cells further expresses the CD138 antigen, whereas all CD38+ plasma cells are also CD138+ in bone marrow. (PMID:11877292, 16956823,18626062).

2.2 Preparation of peripheral blood samples

Blood samples can be obtained in different forms, e.g., as whole blood, buffy coat or buffy cone. It may be necessary to process blood samples upfront to, for example, generate PBMCs for subsequent analysis of peripheral blood B cells. For detailed information, see the MACS Handbook chapter Human  blood.
MACS Handbook:

Blood (human)

2.3 Magnetic separation of B cells from peripheral blood

Miltenyi Biotec has developed numerous products for the straightforward magnetic separation of B cells and distinct B cell subsets. B cells can be isolated either straight from whole blood or buffy coat without density gradient centrifugation or erythrocyte lysis, or from PBMCs after density gradient centrifugation. Both positive selection and depletion strategies can be pursued for direct isolation and isolation of untouched B cells, respectively. 

For details on MACS® Cell Separation Technology, see the MACS Handbook chapter Magnetic cell separation
2.3.1 Isolation of B cells from whole blood, LRSC, or buffy coat

At a glance: Kits and reagents for the separation of B cells from blood

Starting MaterialIsolation strategyCommentsAutomation possible Product
Buffy coatPositive selection of target  cellsBased on CD19 expression.Yes**StraightFrom Buffy Coat CD19 MicroBead Kit, human
Whole blood Positive selection of target  cellsCD19 is expressed on lower levels on plasma cells; for analysis, use CD20. Yes*StraightFrom Whole Blood CD19 MicroBead Kit, human
LRSCPositive selection of target  cellsBased on CD19 expression.Yes**StraightFrom LRSC CD19 MicroBead Kit, human
Whole bloodDepletion of non-target cells

Depletion of activated B cells.

Isolation of B cells directly from whole blood in less than 30 minutes (total capacity: 3x30 mL whole blood).

No MACSxpress Whole Blood B Cell Isolation Kit, human
Whole bloodDepletion of non-target cells

Retains activated CD43+ B cells.

Isolation of B cells directly from whole blood in less than 30 minutes (total capacity: 3x30 mL whole blood).

NoMACSxpress Whole Blood Pan B Cell Isolation Kit, human
Buffy coatDepletion of non-target cells

Retains activated CD43+ B cells.

Isolation of B cells directly from an entire buffy coat in less than 30 minutes.

NoMACSxpress Buffy Coat Pan B Cell Isolation Kit, human
LRSCDepletion of non-target cells

Retains activated CD43+ B cells.

Isolation of B cells directly from LRSC in less than 30 minutes.

NoMACSxpress LRSC Pan B Cell Isolation Kit, human
Whole blood from B-CLL patientsDepletion of non-target cells

For samples from patients with B-CLL.

Isolation of B cells directly from whole blood in less than 30 minutes (total capacity: 3x30 mL whole blood).

No MACSxpress Whole Blood B-CLL Cell Isolation Kit, human
 *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

**Semi- or fully automated high-throughput cell separation with the  MultiMACS™ Cell24 Separator Plus or MultiMACS X.

StraightFrom® MicroBeads allow magnetic cell separation directly from sources such as whole blood, bone marrow, and buffy coat. Pre-enrichment of white blood cells by density gradient centrifugation or other methods, is not required. The purified cells are well-suited for subsequent flow cytometry analysis, molecular biology studies, and functional studies.

StraightFrom Whole Blood CD19 MicroBeads allow the isolation of CD19+ cells directly from 0.25–15 mL whole blood or bone marrow.  Cells in the sample are labeled with Whole Blood MicroBeads and subsequently purified using the autoMACS® Pro Separator, the Whole Blood Column Kit with the MultiMACS™ Cell24 Separator Plus, or manual separators. Similarly effective is the StraightFrom Buffy Coat CD19 MicroBead Kit that enables gentle and fast magnetic isolation of B cells directly from buffy coat. An entire buffy coat from a blood donation of up to 500 mL can be processed in one run. No washing or cell counting is needed; after adding Buffy Coat MicroBeads directly to the sample, labeled cells are separated over Whole Blood Columns using the MultiMACS Cell24 Separator Plus for fast and convenient semi-automated processing, or manually using MACS Separators. If the starting material is LRSC, the StraightFrom LRSC CD19 MicroBead Kit is perfectly suited for fast and automated isolation of CD19+ cells.

Straight From Buffy Coat CD19 MicroBead
Analysis gate
CD19+ cells

CD19+ cells isolated from buffy coat. A buffy coat sample was processed using the StraightFrom Buffy Coat CD19 MicroBead Kit and the MultiMACS Cell24 Separator Plus with the Single-Column Adapter and Whole Blood Columns. Cells were fluorescently stained with CD19-PE, CD20-APC, and CD45-VioBlue® and analyzed by flow cytometry using the MACSQuant Analyzer. Cells were triggered via CD45-VioBlue; cell debris and dead cells were excluded from the analysis based on scatter signals and propidium iodide fluorescence.

Alternatively, the MACSxpress® Cell Isolation Kits allow the isolation of untouched target cells directly from freshly drawn anticoagulated whole blood, buffy coat, or LRSC by depletion of non-target cells. MACSxpress Whole Blood Kits are ideal for the processing of larger sample volumes (total capacity 3x30 mL). Specialized MACSxpress Kits enable the isolation of  B cells from an entire buffy coat or LRSC.

The MACSxpress Whole Blood B Cell Isolation Kit was developed for the isolation of B cells directly from  anticoagulated whole blood without density gradient centrifugation. While non-target cells are removed by immunomagnetic depletion, erythrocytes are sedimented to yield untouched B cells of high purity.

MACSxpress® Whole Blood B Cell Isolation Kit
Unseparated fraction
Unseparated fraction
After separation
After separation

B cells isolated directly from whole blood. Untouched B cells were isolated from 30 mL of human EDTA-anticoagulated whole blood using the MACSxpress Whole Blood B Cell Isolation Kit, a MACSmix™ Tube Rotator, and a MACSxpress Separator. The isolated cells were fluorescently stained with CD45‑VioBlue, CD19-PE, and CD20-FITC and analyzed by flow cytometry using the MACSQuant Analyzer. Non-leukocytes, cell debris, and dead cells were excluded from the analysis based on CD45 expression, scatter signals, and propidium iodide fluorescence.

The MACSxpress Whole Blood Pan B Cell Isolation Kit enables the isolation of untouched B cells including activated CD43+ B cells. 

The CD43 marker is often used for the isolation of untouched B cells by depletion of non-target cells since it is expressed on nearly all lymphocytes but not B cells. However, as CD43 may be up-regulated on B cells upon activation, these activated B cells would be removed as well. The MACSxpress Whole Blood Pan B Cell Isolation Kit avoids depletion of the activated CD43+ B cells.

MACSxpress® Whole Blood Pan B Cell Isolation Kit
Unseparated fraction
Unseparated fraction
After separation
After separation

Untouched pan B cells isolated from 30 mL of human EDTA-anticoagulated whole blood. Blood was processed with the MACSxpress Whole Blood Pan B Cell Isolation Kit, a MACSmix Tube Rotator, and a MACSxpress Separator. The isolated cells were fluorescently stained with CD19-PE and CD45-VioBlue and analyzed by flow cytometry using the MACSQuant Analyzer. Cell debris, non-leukocytes, and dead cells were excluded from the analysis based on CD45 expression, scatter signals, and propidium iodide fluorescence.

Blood samples from patients with B cell chronic lymphocytic leukemia (B-CLL) often have variable and high B cell frequencies, depending on the treatment/disease status of the patient. Thus, Miltenyi Biotec developed the MACSxpress Whole Blood B-CLL Cell Isolation Kit, a specialized kit to isolate untouched B cells from such samples.
MACSxpress® Whole Blood B-CLL Cell Isolation Kit
Unseparated fraction
Unseparated fraction
After separation
After separation

Untouched B-CLL cells isolated from 30 mL of human EDTA-anticoagulated whole blood. Blood was processed with the MACSxpress Whole Blood B-CLL Cell Isolation Kit, a MACSmix Tube Rotator, and a MACSxpress Separator. The isolated cells were fluorescently stained with CD19-PE and CD45-VioBlue, and analyzed by flow cytometry using the MACSQuant Analyzer. Cell debris, non-leukocytes, and dead cells were excluded from the analysis based on CD45 expression, scatter signals, and propidium iodide fluorescence.

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2.3.2 Isolation of lineage B cells from PBMCs

At a glance: Kits and reagents for the separation of B cells from PBMCs

Starting MaterialIsolation strategyCommentsAutomation possible Product
PBMCs Positive selection of target cells For analysis CD20 conjugated antibodies should be usedYes*CD19 MicroBeads, human
Positive selection of target cells and subsequent label removal.

The kit allows the isolation of label-free CD19+ cells, because the complete labeling complex can be released from the cell surface after separation.

For analysis CD20 conjugated antibodies should be used

NoREAlease CD19 MicroBead Kit, human
Positive selection of target cells  Yes*CD20 MicroBeads, human
Positive selection of target cells  Yes*CD22 MicroBeads, human
Depletion of non-target cells Includes activated CD43+ B cells, to isolate untouched B cellsYes*B Cell Isolation Kit II, human
PBMCs from B-CLL patientsIsolation via depletion of non-target cellsSpecifically designed for malignant B cells Yes*B Cell Isolation Kit (B-CLL), human
*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
Instead of working directly with whole blood or buffy coat, the blood samples can be processed over a density gradient centrifugation to pre-enrich peripheral blood mononuclear cells (PBMCs) as starting material for subsequent B cell isolation. 
CD19 MicroBeads, CD20 MicroBeads, and CD22 MicroBeads allow positive selection and depletion of B cells from PBMCs. The lineage markers CD19, 20, and 22 are expressed on all peripheral blood B cells and are down-regulated when the B cells differentiate into plasma cells.
The B Cell Isolation Kit II, human enables the isolation of untouched B cells. The kit contains a cocktail of biotinylated antibodies and Anti-Biotin MicroBeads for magnetic labeling and subsequent depletion of non–B cells.
The B Cell Isolation Kit (B-CLL), human, is designed for the isolation of untouched B cells from tumor samples (fig. 3). During the course of some diseases such as B-CLL, malignant B cells express CD43. The depletion cocktail of the kit has been specifically developed for the isolation of malignant B cells and therefore does not contain a CD43 antibody.
 B-CLL Cell Isolation Kit
Before isolation
After isolation

Isolated untouched B cells containing B-CLL cells. Cryopreserved human PBMCs were processed using the B-CLL Cell Isolation Kit, human. Isolated cells were fluorescently stained with CD19-APC and Anti-Biotin-FITC, and analyzed by flow cytometry.

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2.3.3 Isolation of B cell subsets

The table below gives an overview of different options for isolating B cell subsets from various starting materials using different isolation strategies.

At a glance: Kits and reagents for the separation of B cell subsets

B cell subsetStarting materialIsolation strategyCommentsautomation possibleProduct
Naive B cells whole bloodDepletion of non-target cells NoMACSxpress Naïve B Cell Isolation Kit, human
PBMCsDepletion of non-target cells Yes*Naïve B cell Isolation Kit, human
Memory B cellsPBMCsDepletion of non-target cells and subsequent positive selection of target cellsIncludes switched and IgM positive memory B cells based on common expression of CD27  Yes*Memory B cell Isolation Kit, human
Switched memory B cellsPBMCsDepletion of non-target cells Includes IgG and IgA memory B cells Yes*Switched Memory B cell Isolation Kit, human
IgG+ memory B cellsPBMCsDepletion of non-target cells and subsequent positive selection of target cells Yes*IgG+ Memory B Cell Isolation Kit, human
IgM+ memory B cellsPBMCsDepletion of non-target cells and subsequent positive selection of target cellsIncludes IgM only and IgM+IgD+ memory B cells  Yes*IgM+ Memory B Cell Isolation Kit, human
Plasma cellsPBMCsDepletion of non-target cells and subsequent positive selection of target cells  Yes*Plasma Cell Isolation Kit II, human
 *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.

Naive B cells

The MACSxpress Naive B Cell Isolation Kit, human was developed for the isolation of untouched naive B cells from up to 30 mL anticoagulated whole blood. Non-target cells are removed by immunomagnetic depletion and erythrocytes are sedimented to yield untouched naïve B cells of purity.

If your starting material is PBMCs, use the Naïve B Cell Isolation Kit II, human, which also delivers highly pure naïve B cells by depletion of non-target cells.

Naive B Cell Isolation Kit II, human
Unseparated fraction
Unseparated fraction
After separation
After separation

Untouched naïve B cells isolated from PBMCs. Separation was done with the Naïve B Cell Isolation Kit II, an LS Column, and  MidiMACS™ Separator. Cells were stained with a B cell marker, CD19-FITC, and Anti-IgD-APC and CD27-PE to discriminate naïve B cells (CD19+IgD+) from other B cells.

Memory B cells

The Memory B Cell Isolation Kit, human isolates all memory B cells from human PBMCs. Unwanted cells are depleted followed by positive selection with CD27 MicroBeads.

Miltenyi Biotec also offers solution for the isolation of specific memory B cell subsets. As the kit name implies, the Switched Memory B Cell Isolation Kit isolates all switched memory B cells from human PBMCs, while also depleting IgD and IgM memory B cell.

The IgG+ Memory B Cell Isolation Kit, human and the IgM+ Memory B Cell Isolation Kit, human deliver highly pure fractions of IgG+ and IgM+ memory B cells from human PBMCs, respectively. Isolation is done by depletion of unwanted cells followed by positive selection with Anti-IgG or Anti-IgM MicroBeads.

IgG+ Memory B Cell Isolation Kit
Unseparated fraction
Pre-enriched memory B cells
Isolated IgG+ memory B cells

Specific subsets of memory B cells isolated from PBMCs. IgG+ memory B cells were isolated from human PBMCs using the respective isolation kit. Isolated cells were fluorescently stained  with Anti-IgG-APC and CD19-PE. Cell debris and dead cells were excluded from the analysis based on scatter signals and propidium iodide fluorescence.

IgM+ Memory B Cell Isolation Kit
Unseparated fraction
Pre-enriched memory B cells
Isolated IgG+ memory B cells

Specific subsets of memory B cells isolated from PBMCs. IgM+ memory B cells were isolated from human PBMCs using the respective isolation kit. Isolated cells were fluorescently stained with Anti-IgM-APC and CD27-PE. Cell debris and dead cells were excluded from the analysis based on scatter signals and propidium iodide fluorescence.

Plasma cells

When B cells develop into plasma cells, they downregulate expression of CD19 and CD20 and become negative for CD22. Plasma cells of all differentiation stages are identified by the expression of high levels of CD38. The Plasma Cell Isolation Kit II, human, isolates plasma cells from human PBMCs or bone marrow by depletion of non-plasma cells in a first step. In the second step, pre-enriched plasma cells are directly labeled with CD38 MicroBeads and isolated by positive selection.


Other B cell subset

Other B cell subsets, like activated B cells, can be isolated using a combination of the B cell Isolation Kit II, human (negative selection) with any other MicroBeads for positive selection of a distinctive marker. So, for example, to isolate CD69+ B cells, use the B Cell Isolation Kit II, human followed by CD69 MicroBeads.

2.4 Characterization of B cells by flow cytometry

B Cells are analyzed by flow cytometry based on the presence of different extracellular and intracellular markers. Access an overview of all Miltenyi Biotec Antibodies in the Related Resources panel to the right. 

2.4.1 Flow cytometry panels

At a glance: Markers to clearly distinguish different B cell subtypes

Pan B cellsNaive B cellsPlasma cellsMemory B cellsIgG memory B cellsNatural memory B cellsPost GC cellsRegulatory B cellsActivated B cells
CD19+CD19+CD19dimCD19+CD19+CD19+CD19+CD19+CD19+
CD20+IgD+CD38++CD27+IgG+IgM+IgM+CD24+CD86+
CD45+CD27-CD20dimIgD+CD27+IgD+IgD-CD27+MHCII+
CD38+/-IgD-CD27+CD27+CD38++

Panel for backbone analysis of B cells using the MACSQuant Analyzer

tube 1tube 2tube 3Isotype 1 REA AbsIsotype 2
PECD138IgMIgMmouse IgG1
FITCCD27CD27CD27(IgG1) REAControl S
APCCD24IgGCD86(IgG1) REAControl Smouse IgG1
PE-Vio770CD38CD38CD38(IgG1) REAControl Smouse IgG1
PerCP-Vio7AAD7AAD7AAD
APC-Vio 770CD19CD19CD19(IgG1) REAControl S
VioBlueCD45IgDIgD(IgG1) REAControl S
VioGreenCD20IgAMHCII(IgG1) REAControl Smouse IgG1
2.4.2 Analysis of cytokines

Besides their function as antigen presenting cells (APC) or antibody factories, B cells regulate the immune response by secreting various cytokines. In contrast to cytokine-secreting T cells, however, naïve B cells do not secrete many cytokines upon activation. To become cytokine-producing cells, they require additional stimulation from the surrounding microenvironment or from B differential stages.  Cytokine production in B cells is heterogeneous and dependent on stimuli.  B cells primed by TH1 cells and antigen (Be-1 cells) secrete cytokines associated with type 1 immune responses, such as IFN-γ and IL-12. B cells primed by TH2 cells and antigen (Be-2 cells) make IL-2, IL-13, and IL-4. Regulatory B cells, or B10 cells, are identified by their ability to secrete IL-10 or TGFβ-1. In this way, B cells play a potentially important role in regulating the adaptive immune response before subsequently developing into plasma cells.

Cytokine secretion is measured in different ways. MACS Cytokine Secretion Assays are designed for highly sensitive detection of viable cytokine-secreting cells, allowing detection of secreted cytokines at a single-cell level and simultaneous cell phenotyping. These assays also enable enrichment of viable cytokine-secreting cells.
Cytokines also play a role in class switch determination of the B cell isotype. The following table summarizes the effect of the most important cytokines for class switching.

Summary of cytokine effect on class switching (PMID: 25742773)

IgMIgG1IgG2aIgG2bIgG3IgAIgE
IL-4inhibitinducesinhibitsinduces
IL-6induces
IFN-γinhibitsinhibitsinducesinduces
TGF-βinhibitsinducesinhibitsinduces

2.5 Cell culture of B cells

2.5.1 Expansion of B cells

B cell frequency in human peripheral blood is often too low to obtain sufficient cell numbers for research purposes. Therefore, expansion is an important step in the analysis of B cells, and is usually achieved by stimulation of CD40. The CD40L-CD40 interaction is important in T cell-APC (antigen-presenting cell) interactions and is involved in B cell differentiation and proliferation, isotype class-switching, and protection of B cells from apoptosis, among other functions.  


With the B Cell Expansion Kit, human, you can expand B cells up to 200 times in 14 days. The Kit contains the CD40-Ligand and a cross-linking antibody for multimerization to increase the biological activity of the CD40-Ligand, as well as premium grade Human IL-4 and StemMACS™ HSC Expansion Medium XF, human. B cell expansion is done by restimulation on days 7 and 10 of culture.

B Cell Expansion Kit

Effective expansion of B cells. The components of the Human CD40-Ligand Multimer Kit were pre-incubated 30 minutes at room temperature. B cells were isolated using CD19 MicroBeads and cultured (StemMACS HSC Expansion Media XF supplemented with 5% AB serum and 2 µL IL-4) at a final density of 1×106 cells per mL (high density, A) or 0.15×106 cells per mL (low density, B). On days 7 and 10, cells were harvested, counted, and restimulated. All cells were harvested, counted, and analyzed on day 14. With the high density protocol (A), a B cell expansion of up to 15-fold was achieved. With the low density protocol (B), a B cell expansion of up to 200-fold was achieved.

MACS Handbook:

Cell culture