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Density Gradient Centrifugation for Cell Separation

Updated on Nov 28, 2023

Optimizing a cell separation method is critical for many researchers. There are various ways to sort different types of cells, and the best methodology often depends on the cell subset. One of the quicker and more cost-effective ways to sort a sample based only on physical characteristics is using centrifugation.

What Is Density Gradient Centrifugation?

Centrifugation allows researchers to separate cells based on their shape and size. Samples are placed into a centrifuge—a machine designed to spin liquid solutions at a set speed. The rotation causes the mixture to experience a centrifugal force that pushes larger particles from the center toward the bottom and smaller ones to the top. The larger components experience a stronger centrifugal pull force than the smaller components.

The process is similar to density gradient centrifugation, where samples are also placed into a centrifuge, but the end goal is not to sort them by size. Spinning from the centrifuge causes more dense particles to move to the bottom of the tube because these particles have more mass and are carried further by their inertia. Less dense particles then settle higher within the sample. This process creates a sorted solution layered by particle density from least to most, top to bottom.

Principles of Density Gradient Centrifugation

Each particle has a specific set of physical characteristics; the properties of its biological makeup that can be used for separation and isolation. Density gradient centrifugation focuses on two characteristics—size and density.

The length of time required for this process depends upon the particles’ size. Larger particles will reach their position of stability earlier, whereas smaller particles take longer to pass through the larger particle zone and assume a position deeper in the gradient.

Density Gradient Reagents

In density gradient centrifugation, distinct reagents with specific densities are used to isolate or separate cells. Not only can these products speed up the process, but they can also increase purity and throughput. By keeping particles from clustering—creating a distinct interface—the quality of reagents can greatly increase the efficiency of density gradient centrifugation.

Centrifuge Components

The centrifuge’s spinning mechanism is composed of two distinct parts: the rotor assembly and the electric motor. The electric motor provides the rotation power and the rotor assembly transmits the rotation energy into the rotor tubes. The rotor assembly will hold the density gradient tubes, which are prepped with a media of known density to aid in layer separation.

Density Gradient Media

Density gradient centrifugation uses the natural differences in leukocyte density to separate blood cells into specific cell populations. Using a gradient media of known density aids in developing distinct layers and provides natural media barriers between cell populations.

As the motor spins at very high speeds, the chosen density gradient media are pushed upward by centrifugal force through the whole blood solution. Each media has a set density of its own, such as that used to remove red blood cells from whole blood. In this instance, the density is between that of red blood cells and the PBMCs (human peripheral blood mononuclear cells). This centrifugation-facilitated separation depends on selecting an appropriate medium to divide the sample into the desired populations.

Applications of Density Gradient Centrifugation

Gradient centrifugation is used to purify samples into their component populations. While cell separation is a popular application of density gradient centrifugation, it can also be used to determine the unknown densities of different particles.

Centrifugation of all types benefits researchers because it harvests substances for additional experimentation or medical uses. It helps to remove dead cells and debris in samples so that specific groups of cells or particles may be effectively isolated and studied.

Cell separation methods—such as buoyancy-activated cell sorting (BACS™) or FACS (fluorescence-activated cell sorting)—can be supplemented with centrifugation to increase overall efficiency.

Learn more about how BACS microbubbles can be used with centrifugation to increase productivity.

Differential Centrifugation

Differential centrifugation is another centrifugation separation method that is based on a particle’s mass. Since different-sized cells already behave differently, the process is done with no reagent or medium.

Differential centrifugation is sometimes considered a simpler form of centrifugation. It separates cells and organelles while density gradient centrifugation is used for molecules and particles.

The main difference between the two centrifugation methods is the type of physical properties on which the process is based. Differential centrifugation is more straightforward but density gradient centrifugation can sort much smaller particles for increased specificity. An example of differential centrifugation is the preparation of a buffy coat from whole blood.

Difference Between Density Gradient and Differential Centrifugation

Density gradient centrifugation focuses on separating cells based on their density, rather than mass and size alone, allowing scientists to sort cells that share basic sizes, such as white and red blood cells. While differential centrifugation does not require special separation reagents, density gradients utilize density-known medium reagents to aid in forming distinct cell population layers during the centrifugation process.

Density Gradient Centrifugation and Other Cell Isolation Methods

There are many blood separation techniques, such as flow cytometry and single-cell type isolation available. Flow cytometry utilizes fluorescence, size, and granularity of cell populations to distinguish cells. Flow cytometry is ideal for separating populations of cells based on multiple parameters. Single-cell isolation allows researchers to isolate specific cell subsets. Selecting the right separation technique for different desired final cell populations is determined by evaluating each method’s effectiveness.

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