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).
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.
|Feature||Naive B cells||GC B cells||IgM+IgD+CD27+ cells||IgM-only||IgG+ and IgA+|
|Frequency in peripheral blood||50%||NA||15%||5%||25%|
|Ig isotype expression||IgM, IgD||IgM or IgD||IgM, IgD||IgM||IgG or IgA|
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.
|Starting Material||Isolation strategy||Comments||Automation possible||Product|
|Buffy coat||Positive selection of target cells||Based on CD19 expression||yes*||StraightFrom Buffy Coat CD19 MicroBead Kit, human|
|Whole blood||Positive selection of target cells||CD19 is expressed on lower levels on plasma cells; for analysis, use CD20||yes||StraightFrom Whole Blood CD19 MicroBead Kit, human|
|LRSC||Positive selection of target cells||Based on CD19 expression||yes*||StraightFrom LRSC CD19 MicroBead Kit, human|
|Whole blood||Depletion of non-target cells||Depletion of activated B cells||MACSxpress B Cell Isolation Kit, human|
|Whole blood||Depletion of non-target cells||Retains activated CD34+ B cells||MACSxpress Pan B Cell Isolation Kit, human|
|Whole blood from B-CLL patients||Depletion of non-target cells||For samples from patients with B-CLL||MACSxpress B-CLL Cell Isolation Kit, human|
|* using the MultiMACS™ Cell24 Separator Plus|
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.
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.
B cells isolated directly from whole blood. Untouched B cells were isolated from 30 mL of human EDTA anticoagulated whole blood using the MACSxpress 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.
Untouched pan B cells isolated from 30 mL of human EDTA-anticoagulated whole blood. Blood was processed with the MACSxpress 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.
Untouched B-CLL cells isolated from 30 mL of human EDTA-anticoagulated whole blood. Blood was processed with the MACSxpress 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.
|Starting Material||Isolation strategy||Comments||Automation possible||Product|
|PBMCs||Positive selection of target cells||For analysis CD20 conjugated antibodies should be used||yes||CD19 MicroBeads, 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 cells||yes||B Cell Isolation Kit II, human|
|PBMCs from B-CLL patients||Isolation via depletion of non-target cells||Specifically designed for malignant B cells||yes||B Cell Isolation Kit (B-CLL), human|
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.
The table below gives an overview of different options for isolating B cell subsets from various starting materials using different isolation strategies.
|B cell subset||Starting material||Isolation strategy||Comments||automation possible||Product|
|Naive B cells||whole blood||Depletion of non-target cells||MACSxpress Naïve B Cell Isolation Kit, human|
|PBMCs||Depletion of non-target cells||yes||Naïve B cell Isolation Kit, human|
|Memory B cells||PBMCs||Depletion of non-target cells and subsequent positive selection of target cells||Includes switched and IgM positive memory B cells based on common expression of CD27||yes||Memory B cell Isolation Kit, human|
|Switched memory B cells||PBMCs||Depletion of non-target cells||Includes IgG and IgA memory B cells||yes||Switched Memory B cell Isolation Kit, human|
|IgG+ memory B cells||PBMCs||Depletion of non-target cells and subsequent positive selection of target cells||yes||IgG+ Memory B Cell Isolation Kit, human|
|IgM+ memory B cells||PBMCs||Depletion of non-target cells and subsequent positive selection of target cells||Includes IgM only and IgM+IgD+ memory B cells||yes||IgM+ Memory B Cell Isolation Kit, human|
|Plasma cells||PBMCs||Depletion of non-target cells and subsequent positive selection of target cells||yes||Plasma Cell Isolation Kit II, human|
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.
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.
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.
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.
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.
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 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.
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.
|Pan B cells||Naive B cells||Plasma cells||Memory B cells||IgG memory B cells||Natural memory B cells||Post GC cells||Regulatory B cells||Activated B cells|
|tube 1||tube 2||tube 3||Isotype 1 REA Abs||Isotype 2|
|FITC||CD27||CD27||CD27||(IgG1) REAControl S|
|APC||CD24||IgG||CD86||(IgG1) REAControl S||mouse IgG1|
|PE-Vio770||CD38||CD38||CD38||(IgG1) REAControl S||mouse IgG1|
|APC-Vio 770||CD19||CD19||CD19||(IgG1) REAControl S|
|VioBlue||CD45||IgD||IgD||(IgG1) REAControl S|
|VioGreen||CD20||IgA||MHCII||(IgG1) REAControl S||mouse IgG1|
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.
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.
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.