April 2021 Share
Within the human body, an interconnected system of cells works together to carry out a functional immune response. When presented with a harmful pathogen, a variety of lymphocytes and signaling molecules activate to combat and eradicate the threat. Scientists actively study this process to gain insight into possible routes for immunological research. One way to study the immune response is to look at peripheral blood mononuclear cells (PBMCs).
PBMCs include lymphocytes, monocytes, and any other white blood cell (WBC) with a round nucleus. They are isolated directly from whole blood through the use of different cell separation techniques. PBMCs exist in peripheral blood, where they act as the body’s front line of defense. This requires these cells to have built-in mechanisms to defend the body against disease. Researchers can use their combative behaviors to better understand disease progression, how the body fights off different pathogens, and even to develop advanced therapeutics and treatments.
Studying PBMCs has multiple applications in toxicology research, including:
PBMCs and white blood cells are integral to toxicology research, but they require processing before they can be studied in these ways.
The PBMC isolation protocol involves directly separating lymphocytes from whole blood. Whole blood is the blood that flows through the human body — the raw fluid with no components removed or separated. This is composed primarily of plasma, platelets, red blood cells, and PBMCs. Overall, PBMCs make up a small percentage (about 1%) of a whole blood sample, and are extremely difficult to study when crowded by other substances. PBMC isolation from whole blood must be performed before researchers can perform toxicology experiments.
Three of the most common methods for PBMC isolation include:
Each of these blood separation techniques have their own advantages and disadvantages.
Density-gradient centrifugation relies on physical characteristics such as size and density to sort cell populations. By placing a sample into a centrifuge, a device that can rotate at high speeds, the different cell types will sort themselves out by grouping with particles of a similar density. More dense particles will fall to the bottom or the outside, and less dense particles will stay toward the center or rise to the top. Centrifugation of a standard whole blood sample will separate the general layers of the blood components, concentrating the red blood cells (roughly 45% of total volume) at the bottom of the tube, the buffy coat layer – containing the various white blood cells and platelets (roughly 1% of total volume) in the middle layer, and the blood plasma – containing the watery liquid that allows blood to flow, along with the various proteins and dissolved nutrients & gasses (roughly 55% of total volume) at the top of the tube.
One technique for immune cell isolation involves the use of a flow cytometer, a machine that uses fluorescent light to label different particles based on characteristics such as size, shape, and radiance. This method, called fluorescence activated cell sorting (FACS), allows the sample to flow through a tube that then sorts the components into distinct groups.
FACS requires expensive equipment and properly trained personnel. While this technique can be useful when sorting diverse cell samples that need many different populations sorted, it is incredibly time consuming to use this method for WBC isolation. Typically, the sample will first be “prepared” – or cleaned up – to isolate a specific subset of the original sample’s contents before being processed using flow cytometry. This greatly reduces the time required for cell sorting, as the machine will be processing a much smaller and more concentrated sample volume. It also can result in a larger population of healthy, viable cells at the end, as less natural cell death will occur when processing time is greatly reduced.
Another method binds magnetic beads to target cells and flows the sample through a magnetic field, suspending the cells with magnets attached. Magnetic-activated cell sorting (MACS) is faster and cheaper than the two previously mentioned methods, but it has the highest cell loss of the three. The harsh magnetic fields can rupture cells and cause fractured organelles to clutter the sample. This extracellular debris can cause clumping and blockage which results in a lower throughput.
Buoyancy Activated Cell Sorting, or BACS, leverages the power of gravity to enable cell sorting. This method uses microbubbles that are functionalized to capture analytes of interest and float them to the top of the sample container. The microbubbles are simply mixed into the sample, where they bind to the contaminating cells and float them to the top for removal, leaving the cells of interest isolated at the bottom – untouched and ready for use. The microbubbles carry out their function without any blunt physical forces or harsh magnetic fields, adequately maintaining cell health and physiology for downstream applications.
Akadeum’s RBC Depletion Microbubble Kit offers a quick and easy cleanup to remove residual red blood cells from your WBC-rich PBMC or Buffy Coat preparation. Akadeum’s revolutionary microbubble approach removes up to 99% of RBC contamination in a fast and easy workflow that maintains the health and physiology of delicate cells of interest. Simply mix to bind, spin to separate, and aspirate to discard.
If your process of lymphocyte separation for toxicology research could benefit from the fast, gentle microbubble workflow, be sure to explore Akadeum’s suite of products. We also partner with companies and institutions who are looking to leverage the microbubble platform for next-generation solutions, if you’re interested in learning more about partnering with Akadeum, we’d love to hear from you.
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