Cell separation is the process of extracting a single cell population from a heterogenous biological mixture, such as blood or tissue. Cell separation as a method of sample preparation is a common workflow in life science research. The starting sample can be enriched for the analyte of interest, removing the background noise (like unwanted cells) from the sample. For immunology applications, this can mean enriching the sample for the specific immune cell being analyzed – for example, isolating the B cells contained within the sample for downstream processing.
B cells, or B lymphocytes, are a type of white blood cell that play a vital role in producing antibodies for the human immune response. B cells work together with T cells and the rest of the immune system to combat infections and harmful pathogens in the body. They play an important role in the development of long-term immunity.
Lymphocytes are created in the bone marrow when lymphoid progenitor cells are signaled by bone marrow stromal cells to begin developing B cells.
The process of B cell development can be grouped into two main stages, or phases. The first is the maturation phase, which covers development from hematopoietic stem cell through to the development of the mature naïve B cell. Next is the differentiation phase, which encompasses the antigen-activation of the B cell through to development of antibody-secreting plasma cells and the development of memory B cells.
B cells will change their location within the body throughout their various stages of maturation, with each location providing the appropriate microenvironment for the developmental stage. For example, B cells that are encountering antigens (and receiving help from T cells) form germinal centers with T cells in the follicles, where they undergo selection for B cells with higher affinity receptors. Memory B cells, an important player in long-term immunity, can be found primarily in the spleen.
There are four main types of B cells – transitional, naive, plasma, and memory – that all have their own purpose in the maturation process.
A transitional B cell is the link between immature and mature B cells. While they cannot perform any actions to help fend off harmful pathogens, they do travel between the bone marrow and secondary lymphoid tissues. During this time, they are subjected to checks to confirm they will not produce autoantibodies, or antibodies that attack the host.
Transitional cells are rarely targeted in B cell separation because they do not have as specific of a function as the others. Studying them inside the body is helpful enough.
Naïve B cells are the next step beyond transitional B cells. After the B lymphocyte matures, whether in the bone marrow or secondary lymphoid organs, it will remain a naïve blood cell until it is activated.
Activation occurs when a mature B cell is exposed to the antigen-presenting cells specific to its B cell receptor. Upon activation, a naïve B cell can become a plasma B cell or a memory B cell. Naïve cells don’t fight infection, they simply wait to be activated by a T cell or an antigen-presenting cell (APC).
Naive B cells can be studied, activated, and manipulated with biochemicals that help researchers learn more about the immune system. They have slightly different regulatory markers than the other cells which allows them to be isolated. For example, naive B cells are signaled by CD19+, CD27–, and CD38–.
One important B cell variant is the memory B cell, a type of B lymphocyte that is necessary for building long-term immunities within the body. These cells remain in the bloodstream after an infection subsides. If the host is re-exposed to that same antigen in the future, memory B cells can quickly activate with the help of T cells.
With the extra antibodies created by memory B cells, the immune system can often fend off familiar pathogens before the infection becomes symptomatic. Memory B cells have a CD19+, a CD27+, and a CD38– marker. The different expression on the CD27 marker allows scientists to isolate memory cells and naive cells with only one biomarker.
Plasma B cells, also called effector B cells, are large cells with a very large endoplasmic reticulum (ER). The ER is responsible for helping to synthesize and transport proteins. This composition allows plasma cells to produce large quantities of antigen-specific antibodies.
Plasma cells respond to signal chemicals secreted by T cells during an infection. They continue making antibodies to fight the infection until it is controlled or eliminated. Plasma cells can often be found at the site of chronic inflammation.
Plasma B cells are similar to memory B cells but with a CD38+ biomarker. Because this marker is difficult to target, researchers often sort plasma B cells with FACS and a flow cytometer. With three different lasers, researchers can isolate all three unique cell populations (naive, memory, and plasma) at once with a high throughput. The efficiency of this sort can be dramatically improved by first enriching the sample for B cells (using a sample preparation method like BACS) before sorting the B cells into their subtypes with FACS.
The isolation of B cells from biological samples allows scientists to get a more comprehensive understanding of the body’s immune response to harmful pathogens.. B cell enrichment is a critical step in this workflow, as downstream processing requires that researchers first obtain a large enough population of healthy, viable B cells for analysis. B cell isolation can be performed with multiple methods of cell separation including methods that use buoyant microbubbles, magnetic beads, or fluorescence flow sorting for cell isolation.
Buoyancy activated cell sorting (BACS™) uses buoyant microbubbles to isolate the target of interest. The microbubbles are gently mixed into the sample, where they engage with the targets (in this case, the unwanted and contaminating cells) before isolating those targets through floatation-based separation. The microbubbles, bubble-bound contaminating cells, and subnatant are then removed from the sample container, leaving behind a highly enriched and untouched population of the desired B cells. This fast, easy, and gentle buoyant approach eliminates many of the existing technical hurdles of sample preparation, allowing for single-container processing that enables self-separation. BACS is exceptionally gentle on delicate cells and maintains the health and physiology of B cells during enrichment.
FACS, or fluorescence activated cell sorting, uses a modified form of flow cytometry to analyze and sort cell populations based on their physical characteristics. By running a cell sample through a flow cytometer, researchers can isolate cells based on traits such as size, light scatter, or artificial fluorescent markers. While FACS can be extremely useful for sorting complex heterogeneous mixtures, the machinery required to perform this process is very expensive, and a flow sort can be time-intensive if the sample has not been sufficiently enriched to eliminate background signals prior to sorting.
When trying to isolate multiple B cell subtypes from a larger population, FACS allows the researcher to sort B cells into different groups according to cellular characteristics. This process can be further optimized by first enriching the sample for B cells prior to sorting using a method like Buoyancy-Activated Cell Sorting (BACS™), which can decrease sort times as much as 15-fold and help to obtain larger, healthy populations of rare cell subtypes.
Magnetic bead-based cell sorting uses magnetic microbeads to bind to target cells, then exposes the sample to a magnetic field. The cells bound to the magnetic beads are held by a rare earth magnet, while the remainder of the sample is removed. The bound cells are then discarded (if using a negative selection protocol to remove unwanted populations) or collected for downstream use (if using a positive selection protocol to collect the wanted cells). While magnetic bead-based enrichment is a commonly used workflow, the harsh magnetic field can be harmful to fragile cell populations. Prolonged exposure to these forces could cause cells to rupture, releasing extracellular debris that can contaminate the sample. In some cases, there are also reports of cellular uptake of the magnetic beads, further complicating research requiring isolation of rare and delicate immune cells.
Mice are frequently used in research as a model of human disease response. Because human and murine B cells are similar in how they are engaged to fend of harmful pathogens, researchers will often test hypotheses with murine B cells before moving to human cells because they are accessible and can be directionally indicative of what a human B cell response might look like. In mice, B lymphocytes are found in high concentrations within the spleen. For this reason, mouse splenocytes are a common sample type used for B cell analysis.
When enriching mouse splenocyte samples for B cells, the ultimate goal is to isolate the largest possible population of healthy, viable B cells that can be used for further downstream analysis. These cells can include numerous B cell subtypes, so an initial cleanup step to enrich the sample for B cells prior to further processing (like FACS) can help to speed up sort times, decrease background noise, and ultimately create a highly-enriched population of healthy, viable cells of interest.
Akadeum’s Mouse B Cell Isolation Kit delivers a highly enriched population of healthy, viable B cells using a protocol that is fast, easy, and gentle on cells. Ideal for enrichment of delicate immune cells, Akadeum’s novel Buoyancy Activated Cell Separation (BACS™) occurs directly in the sample container without exposing cells to harsh chemicals or harmful shear forces. The Akadeum microbubble enrichment protocol enables quick and easy sample preparation that takes about 30 minutes from start to finish without additional equipment or expensive consumables like magnets or columns.
The end result is a high-purity population of mouse B cells that are ready for downstream use – in fact, using prior to cell sorting can greatly reduce sort times, saving time and money while delivering a highly viable final population.
Akadeum’s microbubble approach to B cell enrichment allows researchers to obtain highly enriched, high-quality B cells using our exceptionally gentle workflow that does not sacrifice purity for quality, as the negative selection microbubble process maintains cell health and physiology. Laboratories around the country are using microbubbles in their novel and ground-breaking research with exceptional results. Not only does Akadeum’s kit result in a pure, enriched population of B cells, but the microbubble workflow allows the isolated B cells to retain their activation and differentiation potential, and the enrichment process does not disturb B cell receptors (BCRs) or skew the isolated population.
• Ichwaku Rastogi, a researcher with the Doug McNeel Lab at the University of Wisconsin, found that using Akadeum’s Mouse B Cell Isolation Kit delivered a highly enriched, healthy B cell population that retained its antigen-presenting capabilities.
• Derek Jones at the University of Pennsylvania found that the kit delivered a high-purity, healthy B cell population that was not activated by the isolation process and that maintained the ability to be differentiated into plasma cells.
• A research group from the University of California San Francisco found that the kit created an enriched B cell population, and the isolation protocol did not disturb the B cell receptors (BCRs) or skew the isolated population.
Akadeum’s microbubble technology is the next generation in isolation techniques. They have been developed to meet the demand for a more efficient platform with higher throughput capabilities and improved scalability. Microbubbles offer a simplified workflow that doesn’t require any extra equipment, costly consumables, or specialized training. The easy-to-follow protocol requires limited handling which enables processing multiple samples at a time, with enrichment occurring directly in the sample container eliminating the need for multiple tube transfers. The fast, easy, and exceptionally gentle microbubble workflow yields highly viable cells with unaltered physiologies.
Interested in experiencing the microbubble difference for yourself?
If you have any questions or would like to speak to one of our scientists directly about your work to determine if there’s an application for Akadeum’s microbubbles, please get in touch today! We would love to hear more about your cell separation needs and help you to develop a microbubble workflow that overcomes common headaches in sample preparation.