Similar to humans, mice have complex immune systems made up of innate and adaptive immune cells. While innate cells act as the first line of defense by eliciting a general, non-specific response against pathogens, adaptive immune cells generate a more complex anti-pathogen response that is antigen-specific.
Mouse B cells are essential components of the adaptive immune system, as they are primarily responsible for generating antibody-mediated immunity. These lymphocytes can be generally characterized by the presence of antigen-specific immunoglobulin (Ig) receptors on their cell surface. Functional rearrangement of the Ig loci yields a highly diverse set of antigenic specificities that allow immunoglobulins to bind foreign antigens and facilitate pathogen destruction.
Aside from Ig-mediated functions, B cells support the immune system in many other ways. Key cytokines secreted by B cells are essential for immune cell development and homeostasis—particularly for T cell and dendritic cell activity. Regulatory B cells help balance inflammatory responses, and B cells impact the organization of lymphoid tissue, among other functions. B cells are integral for balanced immune function.
Murine B cell maturation begins in the bone marrow. The first steps of development in B cell progenitors, also known as pro-B cells, are characterized by the expression of a semi-functional precursor B cell receptor (pre-BCR). Upon completion of this step, pre-BCR-bearing cells proliferate and differentiate into immature/transitional B cells that express Immunoglobulin M (IgM) on their surface.
Immature B cells leave the bone marrow and travel to the spleen for further development. In the spleen, differentiation continues as B cells undergo a BCR refinement process resulting in a repertoire of non-self-reactive, immune-competent naive B cells. Naive B cells enter the periphery and circulate throughout the body via the bloodstream or migrate to secondary lymphoid organs, awaiting activation through encounters with their cognate antigen.
Murine B cells are classified into subsets based on their developmental location and function. The principal classes of B cells—B-1 and B-2 cells—develop from fetal hematopoietic stem cells and bone marrow-derived precursors, respectively. Further, B-1 cells perform innate cell-like functions and B-2 cells are progenitors to more terminally differentiated effector cells, like plasma and memory cells.
B1-B cells are the first murine B cells to develop, deriving from embryonic and fetal progenitors. B-1 B cells promote the establishment of a natural antibody repertoire by producing natural antibodies, antibodies found in the circulation of normal individuals that are not the result of antigen exposure. These natural antibodies are specific for non-protein antigens, usually lipids, and are important for combating microbial infections.
B-2 B cells are a subset of murine B cells primarily derived from bone marrow that elicit more pathogen-specific antibody responses. Once out of the bone marrow, B-2 cells migrate to the periphery and secondary lymphoid organs, giving rise to follicular and marginal zone B cells.
Follicular B cells make up the majority of circulating B cells and serve as precursors for more developmentally mature B cell subsets, including plasma and memory cells. Murine marginal zone B cells reside in the marginal sinus of the spleen. It is here that B-2 cells work together with T cells to generate high-affinity, long-lasting antibody responses.
Regulatory B cells, or Bregs, act as negative regulators of the immune system to dampen pathological immune responses. Breg activity is crucial for supporting healthy immune function—too few of these cells can lead to rampant inflammation, resulting in autoimmune diseases. Bregs primarily exert their immunosuppressive function via secretion of the cytokine IL-10.
Upon exposure to antigen, naive B cells become activated and traffic to the germinal centers, where many of them will undergo the germinal center reaction. This kicks off a series of genetic rearrangements to increase the affinity of the antibodies they produce and begin the process of isotype switching. This results in these cells to terminally differentiate into two effector subsets: plasma cells and memory B cells. Murine memory B cells are a subset of long-lived murine B cells that circulate and provide quick responses to future exposures to their specific antigen, thus contributing to immunologic memory.
There are two types of plasma cells. Short-lived plasma cells are terminally differentiated antibody-secreting cells generated in response to T independent antigens. Long-lived plasma cells are generated through marginal zone reactions and migrate to the bone marrow where they continue to produce antibodies against their specific antigen. Approximately half of the antibody in circulation in an adult is produced by long-lived plasma cells targeting previously encountered antigens.
Cell markers expressed on the surface of B cells can be used to identify cells at various stages of development. Conventionally, all B cells share expression of the marker CD19, the pan B cell marker.
Mature B cells additionally express CD20 and CD22. While not entirely exclusive to B cells, the marker B220 (CD45R) is typically used alongside other surface markers to identify B cells, as it is essential for B cell receptor signaling. Additional standard markers include CD138, found on plasma cells, and CD23, typically found on activated B cells.
As subsets of B cells undergo differentiation, the presence or absence of different Ig isotypes can be used in conjunction with the expression of unique cell surface markers for further characterization.
The presence of early Ig isotypes, such as immunoglobulin M and immunoglobulin D, can help identify murine lymphocytes that are early in B cell development—such as transitional B cells or those with innate-like defense functions (i.e., marginal zone B cells). The absence of these Ig isotypes can, likewise, identify B cells that are more developed, such as follicular, plasma, and B cells located in germinal centers.
Peripheral blood, bone marrow, spleen, and other secondary lymphoid tissues, like gut-associated lymphoid tissue (GALT), can all serve as sites for B cell extraction. Bone marrow and spleen are considered especially prime locations due to their high concentrations of murine B cells. Extraction of marrow from murine bones and splenocytes from homogenized murine spleens can serve as excellent sources of cell populations from which to isolate B cells via appropriate methods
Cell isolation is the process of separating a particular subset of cells from the heterogenous pool of cells in the body, typically using expressed surface markers to extract cells of interest. The end result is a homogenous sample of a single cell type.
B cells can be isolated from other unwanted immune cells using unique cell surface markers, such as CD19, for use in downstream in vitro and in vivo applications. In many cases, it is imperative to isolate a uniformly pure collection of cells given that cell-cell interactions can alter B cell activity, especially in the case of naive B cells.
Numerous methods can be employed to isolate murine B cells, including fluorescence-activated cell sorting (FACS), magnetic-activated cell sorting (MACS), and density gradient centrifugation. Method selection should factor in how cells will be used post-isolation. It is important to consider how B cell integrity, function, and quality may be impacted.
BACS, or buoyancy-activated cell sorting, is a novel approach by Akadeum to isolate mouse B cells using buoyant microbubbles. Antibody cocktails containing antibodies specific for non-B cell antigens are mixed with heterogeneous samples to label the unwanted cells. Microbubbles are added and bind to these unwanted cells, leaving the B cells untouched.
The unwanted cells float to the top of the sample, away from the cells of interest, due to the microbubbles’ intrinsic buoyant properties. Finally, the unwanted cell-bound microbubbles are removed, leaving a homogenous, high-purity sample of B cells.
Compared to other methods like MACS (magnetic-activated cell sorting) and FACS (fluorescence-activated cell sorting), BACS is gentler and much more cost-effective. Potentially damaging magnetic fields and iron beads are not required, and expensive cell processing equipment is not necessary to isolate target cell populations.
BACS relies on one simple factor—the principle of buoyancy. This means BACS can be performed at any scale, in any size laboratory space, with no need for bench space to fit a fluorescent cell sorter, and without extensive training or no highly skilled personnel required.
View our product page to learn more about Akadeum’s Mouse B Cell Isolation Kit.