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Counterflow Centrifugation

Updated on Aug 21, 2023

Scientists working in the laboratory

What Is Counterflow Centrifugation Elutriation?

Centrifuges are one of the most commonly utilized laboratory instruments, applicable for cell separation, purification, and harvesting. Counterflow centrifugation elutriation (CCE) is a liquid clarification technique that separates cells suspended in a solution by size and stage. This process operates based on the differing sedimentation rates of cells or particles of different sizes in a liquid medium under centrifugal force. 

CCE is a valuable method for separating cells of a specific size or cell density in a heterogeneous cell population. Commonly implemented in research settings for purifying cell populations for downstream applications, counterflow centrifugation has become a staple in sample preparation.

How Does Counterflow Centrifugation Work?

Counterflow centrifugation takes its name from the opposing forces within and around the instrument’s chamber as they flow against each other. CCE involves passing a cell suspension through a chamber containing a dense liquid medium, flowing at a constant and controlled rate. As the cells flow through the chamber, smaller particles are pushed out of the compartment and larger particles are kept within the circulating liquid medium. 

The buffer’s constant flow rate is integral to this method’s underlying physics and functionality. The inner chamber increases rotational speed against the constant flow of the liquid, resulting in a gradual increase in sedimentation rate. By maintaining a constant flow rate of buffer and an opposing centrifugal force, the density of the chamber increases. 

As the cells in suspension move outward within the chamber, the cells or particles are exposed to changing centrifugal force proportional to the distance from the chamber’s center. The cells separate according to their sedimentation properties. Larger particles sediment more rapidly than smaller particles and are collected at a higher centrifugal force and can be collected in fractions as they exit the chamber. The separated cells are pushed from the chamber by size and density, which is useful for isolating various cell populations. 

Akadeum’s Technology is Compatible with Counterflow Centrifugation Protocols

Akadeum’s straightforward and powerful counterflow centrifugation protocol prepares your sample for use with our gentle cell-sorting microbubbles. 

Access robust populations of isolated cells with the simple addition of Akadeum’s buoyancy-activated cell sorting (BACS™) microbubbles to your cell suspension and centrifuge. Compatible with instruments, such as the Thermo Fisher Scientific Rotea or Cytiva Sepax for blood separation with a centrifuge, Cell washing upstream of Akadeum’s cell separation is ideal for cell isolation from leukopak material. 

Advantages of Counterflow Centrifugation

CCE offers many advantages to the downstream analysis of cell populations, including improved separation and purification. Counterflow centrifugation allows for the sorting of cells based on density, which leads to purer populations. In addition to precision, CCE gently sorts cells without harsh chemicals, leaving the population ready for downstream applications. 

Counterflow centrifugation is highly precise and flexible, capable of separating small or large particles across a significant range, increasing its versatility in research initiatives. It’s also relatively quick, delivering rapid separation of large amounts of cells in a short amount of time. 

Disadvantages of Counterflow Centrifugation

Although rapid, CCE does have disadvantages, including limited throughput. Despite its speed, the maximum throughput of CCE is limited by the size of the centrifuge rotors and the time allotted for separation, presenting a challenge for very large sample sizes. 

Achieving the optimal counterflow rate required for CCE can be challenging and time-intensive due to the demands of a continuous flow system. This elaborate design can only replicate with the proper equipment. 

CCE is a versatile and rapid solution for separating cells by size but is limited in other separation procedures unrelated to size or density, even in cases where other characteristics, such as surface protein markers, are more relevant. 

Using Counterflow Centrifugation to Wash Cells for Leukopak Isolation

After peripheral blood mononuclear cells (PBMCs) are collected during leukapheresis, the leukopak needs to be washed and processed for downstream use. CCE is an alternative to density gradient centrifugation, wherein the PBMCs are separated from red blood cells and granulocyte contamination, developing into a distinct layer based on cell density. 

The Benefits of Akadeum’s Products for CCE

Explore how Akadeum’s innovative microbubble technology can improve cell separation and sample preparation with ease. When paired with BACS, counterflow centrifugation elutriation will yield a strong and pure collection of white blood cells viable for research or medical applications. 

Save significant time and protect fragile cells of interest by implementing Akadeum products downstream of CCE to produce quality isolated cell populations every time. 

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