Positive Vs. Negative Selection of Target Cells: Differences Between Positive and Negative Selection

The ability to isolate and separate specific cell populations is critical to scientists looking to understand immune responses, disease mechanisms, and therapeutic interventions. Two fundamental techniques—positive selection and negative selection—serve as the basis for effective cell isolation.

What’s the Difference Between Positive and Negative Selection?

Positive Selection: Tailored Precision and Purity

Positive selection is a collection of strategic approaches employed in cell isolation protocols to enrich and retrieve target cells from a larger, heterogeneous population. These techniques target the desired cell population using antibodies specific to biological markers on the cell’s surface.

By conjugating the antibodies to magnetic beads, nanoparticles, or other support molecules, researchers can utilize affinity columns or magnetic columns to capture the cells of interest. The targeted cells remain for downstream analysis that may involve further cell sorting techniques.

Imagine a scenario where a researcher aims to isolate CD4+ T cells with high purity from a mixed population of immune cells for downstream genome sequencing. They could use antibodies specific to the CD4 marker for positive selection, effectively labeling the target T cells. In this example, cells would be labeled using magnetic particles coated with the CD4-specific antibody. The cell mixture would then be passed through a paramagnetic column resting within a magnetic field, where these particles (and the CD4-expressing cells they are attached to) would become trapped. The cells that are not labeled with magnetic particles would pass through the column freely. The column is then removed from the magnetic field, and the previously trapped magnetic particles can now flow through the column into a fresh collection vessel below. This positive selection enables precise enrichment of the desired cell type.

Negative Selection: Averting Contaminants and Preserving Functionality

Conversely, negative selection isolates cells by removing unwanted cells while leaving the target population untouched. This technique is especially useful when target cells lack exclusive surface markers, as often happens with rare or less-characterized cell types. It is also the preferred technique when the functionality of the cells must be preserved.

In most cases, surface markers that are unique to a specific cell type also have a function that is specific to that cell’s role. These markers can be used to identify these cells because they are not normally expressed on other cell types. Therefore, negative selection can employ antibodies specific to the undesired cells, allowing for their removal from the sample while maintaining the functionality of the desired cells that remain.

Consider a scientist who needs to isolate monocytes from a mixture of immune cells, such as peripheral blood mononuclear cells, for downstream ex vivo maturation and expansion. Antibodies specific to markers expressed on unwanted lymphocytes and granulocytes can be utilized to remove them. By conjugating these antibodies to a sorting molecule, such as a buoyant microbubble, lymphocytes and granulocytes will bind and float to the surface where they can be removed, leaving the untouched monocytes behind for downstream experiments.

Studies like the recent work of Hornschuh et al. demonstrate that negative selection techniques are preferred to maintain monocyte viability and performance after separation. Negative selection ensures the functionality of the isolated monocyte population, which is essential for downstream clinical applications.

Advantages and Disadvantages of Positive vs. Negative Selection

The main advantage of positive selection is that isolated cells are highly purified compared to negative selection. This high purity is due to the use of specific antibodies to singularly target a particular cell type. Additionally, sequential isolations can be performed on the negative cell fraction to further select and purify other desired cells. However, these techniques mean the positively selected cells are bound to the antibodies or other labeling agents, which can affect some downstream assays and the overall cell functionality.

Negative selection, on the other hand, produces targeted cells that are unbound by antibodies, preserving their viability and functionality. The major disadvantage of negative selection methods is that the resulting enrichment is inherently less homogenous than positive selection methods. Targeting all unwanted cells is more difficult than targeting one desired cell population to retain. This can be a problem when the sample contains unknown cell types. However, if the cell population is well characterized, negative selection techniques are typically preferred, especially if the isolated cells are for use in downstream functional assays or clinical research applications.

The Selection Toolkit

Whether positive or negative, the choice of antigens and binding molecules is pivotal for successful cell enrichment. A variety of targets are harnessed to isolate cells of interest, each catering to specific cell types and research goals. Some of the most popular techniques and targets showcase their significance and versatility:

Streptavidin/Biotin System: A Versatile Duo

The streptavidin/biotin system is a widely embraced cell isolation technique due to its versatility and high affinity binding. Streptavidin, a protein derived from Streptomyces avidinii bacteria, binds strongly to biotin, a vitamin B7 derivative. This interaction forms the basis for labeling cells with biotinylated antibodies.

Researchers can conjugate biotin to antibodies specific for their target cells, creating a bridge between the antibodies and streptavidin-coated surfaces. Streptavidin-coated beads or plates then capture the biotin-labeled cells, facilitating selection. This system’s adaptability allows for isolating a wide range of cell types by customizing the choice of biotinylated antibodies.

Red Blood Cell Antigens: A Hematological Approach

Red blood cell (RBC) antigens, well-studied because of their role in blood typing, can also be employed in cell selection. Researchers can target RBC antigens, such as ABO or Rh antigens, with antibodies. These antigens are commonly utilized in negative selection protocols to efficiently remove contaminating RBCs from samples, leaving the desired cells untouched.

Targeting B Cell Antibodies: Exploring Immune Diversity

For B cell isolation, antibodies against specific B cell markers are central to both positive and negative selection approaches. Antibodies against CD19 are commonly used in protocols to isolate B cells for various applications, including immunological studies and therapeutic development. Positive selection with these antibodies allows for precise enrichment of B cells, while negative selection to isolate B cells would use antibodies targeting cell type-specific markers not expressed on B cells to isolate them from other immune cell types.

Fine-Tuning With T Cell Antibodies

Isolating CD4+ and CD8+ T cells, crucial players in the immune response, often involves the use of antibodies against the CD4 or CD8 coreceptors. In positive selection, biotinylated anti-CD4 or anti-CD8 antibodies can be paired with streptavidin-coated surfaces to capture T cells specifically.

Conversely, negative selection can be achieved by employing antibodies against a specific T cell type. For example, CD8 can be targeted to remove unwanted T cell subsets while preserving CD4+ T cells.

How Do I Choose a Cell Separation Approach?

The choice to use a positive or negative selection method depends on your cell population of interest and potential research applications. Both techniques play vital roles in enabling researchers to dissect complex cell populations, investigate immune responses, and develop therapies.

Cells with a robust selection marker on the surface of the target cell type will perform well with positive selection methods. Negative selection methods should be considered for cells with weak or unknown markers. Additionally, if antibodies or other labeling agents attached to targeted cells will affect downstream assays or research applications, it may make more sense to use negative selection methods.

Applications that require negative selection would benefit from buoyancy-activated cell sorting (BACS™) techniques. BACS™ is Akadeum’s cell separation technology that involves sorting cells with buoyant microbubbles. For negative selection, the microbubbles are coated with molecules that bind to target cells, lifting them to the solution’s surface. Once the cells reach the top, they can be removed from the sample via vacuum aspiration, leaving behind an enriched sample at the bottom.

Microbubbles allow researchers to scale their experiments and expand their diagnostic strategies to rare cell populations. This innovative method can be customized to target many different cell-specific biologic analytes while maintaining high recovery, purity, yield, and viability.

BACS™ is fast, easy, and inexpensive compared to other methods, and it preserves cell health and physiology for downstream applications. It can be used in conjunction with other techniques to purify a sample further or by itself as a standalone isolation method.

Akadeum’s BACS™ Technology: Revolutionizing Cell Isolation

Positive selection and negative selection techniques serve as indispensable tools in the microbiologist’s toolkit, facilitating the isolation and separation of specific cell populations with precision and purity. In the ever-evolving cell biology landscape, Akadeum offers cutting-edge solutions and products to enhance cell isolation workflows.

Through microbubble-based cell isolation technology, Akadeum provides researchers with a powerful tool to streamline selection protocols. By leveraging microbubbles coated with antibodies tailored to their application, researchers can achieve rapid and efficient isolation of target cells with unparalleled precision.