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Introduction

Cell biology research is complex, given the many variables that researchers must be aware of and account for to obtain meaningful results. Conducting experiments on an isolated population of cells, rather than a heterogeneous mixture of cells, is a common approach to reduce experimental complexity. This allows cell biologists to confidently attribute observed effects and responses to a particular cell type. Thus, mastering the basic techniques of cell isolation is a valuable skill for any cell biologist.

What is cell separation?

Cell separation, also commonly referred to as cell isolation or cell sorting, is a process to isolate one or more specific cell populations from a heterogeneous mixture of cells. There are a number of cell separation methods available, each with its own pros and cons.

Why do scientists isolate cells?

Conducting experiments on isolated cells allows scientists to confidently answer specific research questions by minimizing interference from other cell types within the sample. Isolated cells have many applications within life science research, such as allowing scientists to:

• Conduct molecular analysis of a single cell type, such as RNA expression and epigenetic analysis
• Genetically modify and expand a particular cell type of interest for disease modeling or cell therapy research applications (e.g. T cell therapy research)
• Directly use purified cells for adoptive cell transfer experiments in various animal models
• Increase sensitivity of analytical methods (e.g. cell isolation for HLA analysis, cell isolation for FISH analysis)
• Study the in vitro effects of drug candidates on specific cell populations (e.g. hematotoxicity testing)
• Fuse enriched plasma cells with myeloma cells to produce hybridomas

How do scientists prepare samples for cell separation?

There are many different ways to prepare samples for optimal cell isolation. The method you select depends on your starting sample and may involve removing certain elements from it or simply creating a single-cell suspension.

Cell separation can be performed on a variety of complex biological samples, including:

• Whole peripheral blood
• Leukapheresis products (e.g. Leukopaks)
• Peripheral blood mononuclear cells (PBMCs)
• Bone marrow
• Cord blood

See page 33 for more information on isolating cells from tissue and page 28 for isolating cells from blood.

Cell Separation Methods

There are multiple methods that can be used to perform cell isolation. Below, we will discuss several cell separation techniques, including their advantages and limitations.

The cell separation method you choose typically depends on what you intend to use the isolated cells for, and the choice may involve a trade-off. For example, if you need very pure cells, you will likely choose a method with high purity but that may result in lower yield.

Immunomagnetic Cell Separation

Immunomagnetic cell separation is a technique whereby magnetic particles are used to isolate target cells from heterogeneous mixtures. To accomplish this, the magnetic particles are bound to specific cell surface proteins on the target cells via antibodies, enzymes, lectins, or streptavidin. The sample is then placed in an electromagnetic field that pulls on the magnetic particles, bringing the labeled cells with them. The unlabeled cells remain in the supernatant, thus creating a physical separation between target and non-target cells within the sample.

Due to its speed and simplicity, immunomagnetic cell separation is one of the most commonly used methods used by scientists to isolate highly purified populations of specific cell subsets. Immunomagnetic cell separation has several advantages, including:

• High purity
• Fast protocols
• Ease of use
• Low equipment cost
• High throughput
• Potential for automation
• High cell viability

Both positive and negative selection can be performed using magnetic cell isolation methods. When a positive selection is performed, the supernatant can be discarded and the magnetically-labeled cells of interest remain immobilized until removed from the electromagnetic field. When a negative selection is performed, the desired cells are located in the supernatant.

Antibodies For Phenotyping And Purity Assessments

Choose from a wide range of antibodies against human or mouse antigens that are verified to work with �����ƽ��’s cell isolation kits in specific downstream applications such as flow cytometry.

Immunomagnetic cell separation is a much faster and simpler procedure than FACS and is often the preferred cell isolation method for common cell types. FACS has several advantages over immunomagnetic cell separation, including the ability to:

• Sort single cells
• Isolate cells based on intracellular markers (e.g. GFP)
• Isolate cells based on surface marker expression levels
• Sort complex cell types with multiple markers at higher purity

Density Gradient Centrifugation

Density gradient centrifugation relies on the varying densities of cells within a heterogeneous sample. The sample is layered on top of a density gradient medium before being centrifuged. During centrifugation, each cell type will sediment to its isopycnic point, which is the place in the medium gradient where the density of the cells and medium are equal.

Common applications of density gradient centrifugation include the fractionation of peripheral blood mononuclear cells, exclusion of dead cells from a cell culture, and separation of plasma from blood cells.

There are several types of density gradient media, each with unique properties that render it ideal for specific purposes. The following are examples of the most well-known types:

• Lymphoprep™, Lympholyte®, and Ficoll-Paque® are similar media that consist of saccharides and sodium diatrizoate; they have a density of 1.077 g/mL. These media are commonly used to isolate mononuclear cells from peripheral blood, cord blood, and bone marrow.
• Percoll® (density: 1.131 g/mL) consists of colloidal silica particles coated with polyvinylpyrrolidone (PVP) and is widely used to separate cells, organelles, viruses, and other subcellular particles.
• OptiPrep™ is a medium consisting of iodixanol in water that is used to isolate viruses, organelles, macromolecules, and cells.

Should I use magnetic cell separation or FACS to isolate cells?

Magnetic cell separation and fluorescence-activated cell sorting (FACS) are the two most common ways by which scientists isolate specific cell types. The choice between the two methods depends on what you require for your specific downstream application.

Magnetic cell isolation is a much faster and simpler procedure than FACS and is often the preferred cell isolation method for common cell types. However, unlike magnetic cell isolation, FACS will allow you to:

• Sort single cells
• Sort multiple cell types simultaneously
• Isolate cells based on intracellular markers (e.g. GFP)
• Isolate cells based on expression levels
• Sort complex cell types with multiple markers at higher purity

To decide which of the two methods to use, start by investigating whether the expected purity of available magnetic cell isolation kits would meet your experimental needs. Product performance data can often be found on a supplier’s website. If a vendor does not publicly provide performance data for their cell separation products, contact them directly to ask for this information or ask for a sample of their product to test in your own lab. Due to their speed and simplicity, magnetic cell isolation techniques can often be easier to incorporate into your experimental design than complicated flow sorting instruments and protocols.

Magnetic cell separation techniques and FACS can also be used together. Pre-enriching your sample with magnetic cell separation techniques prior to FACS can maximize yield and purity and reduce sort time, especially when working with large sample volumes or rare cell types.

What is the difference between positive selection and negative selection?

Positive selection immunomagnetic cell separation methods involve directly labeling desired cells for selection with an antibody or ligand that targets a specific cell surface protein. The antibody or ligand is linked to a magnetic particle, causing the labeled, desired cells to be retained in the tube when the sample is incubated in a magnetic field and the supernatant is discarded. Typical features of positive magnetic selection methods include:

• High purity of isolated cells
• Isolated cells that are usually bound by antibodies and magnetic particles
• An antibody cocktail that targets a unique surface marker on the target cells
• Possible sequential isolation of cell populations from the negative fraction