November 2022 Share
Peripheral blood circulates within the human body carrying nutrients, chemical messengers, water, and a wide variety of cell types. An array of bone marrow-derived hematopoietic cells – white blood cells and red blood cells – distribute into all areas of the body to defend against invaders and carry oxygen, respectively
The most common clinical use for whole blood involves trauma or serious injury in which the patient has lost significant amounts of blood. Blood from the donor is transfused into the patient to replace what they have lost. Peripheral blood can also be separated into its components, such as red blood cells (RBCs) and human peripheral blood mononuclear cells (PBMCs) to be further used in treatment and research. Whole blood is the primary source of immune cells used in a wide range of research applications.
In many research applications, it is necessary to access high-concentration samples of specific cell types to understand the triggers and underlying mechanisms of the human immune system. Whole blood can be separated into these components using various cell isolation or blood separation approaches. For example, leukopaks can be utilized as a PBMC-rich single-donor source of various leukocytes of interest.
Scientists can target distinct cell types within a mixture of blood components using Akadeum’s microbubbles. By utilizing specially formulated antibody cocktails, our microbubbles will bind directly to the unwanted cells within a sample and isolate the target population via negative selection. Using Akadeum’s Human T Cell Isolation Kits, all non-T cell blood cells are tagged with antibodies and simply lifted away from the precious T cells. Learn more about targeted negative selection and how it leaves desired cell populations untouched.
PBMCs originate within the bone marrow and circulate within peripheral blood. These cells make up the majority of the body’s immune response and can be a source of insight into the human immune system and immunologic memory.
PBMCs—or agranulocytes—are divided into cell subsets: monocytes, dendritic cells, and lymphocytes. Agranulocytes are leukocytes—white blood cells—that do not contain granules in their cytoplasm.
Monocytes are a subset of white blood cells that find and destroy pathogens, such as bacteria or viruses in order to stop infection. They reside within tissues and traffic throughout the body within the bloodstream until a possible pathogen activates them.
Dendritic cells are specialized white blood cells that are found within many of the body’s tissues. They possess the ability to process both internally- and externally-derived antigens on major histocompatibility complexes on their surface to alert other immune system cells to the threat. They are a key part of the innate immune system but also serve an important role in the human adaptive immune response.
T cells, B cells, and natural killer cells – NK cells – make up the subset of white blood cells called lymphocytes. The function of these cells to control the specificity of certain immune responses is a highly studied and valuable phenomenon.
B and T cells play an integral role in adaptive immune responses, recognizing pathogen-derived antigens within the bloodstream and tissues. Upon activation, they produce a flood of secreted and cell surface effector proteins in order to destroy the threat and clear the infection. Residual memory T cells and B cell-produced antibodies will remain in the bloodstream to defend against the immune system being exposed to the same threat again.
While all blood cells, including PBMCs, are found in peripheral whole blood samples, other blood products make better starting materials for researchers due to the concentrations of various blood components. Isolating PBMCs from whole blood is time-consuming for relatively low yield and is difficult to scale for research applications.
A buffy coat is a widely used first-step separation product with a higher PBMC concentration than whole blood. The generation of a buffy coat takes advantage of the specific density differences among the various cell types within blood. During centrifugation, the cells form natural layers. The buffy coat refers to the PBMC-rich middle layer of the sample.
Leukopaks present a highly concentrated PBMC source that is commercially available. Collected via leukapheresis, leukopaks also provide large single-donor samples. This reduces the donor-to-donor variability found in other methods. The number of hematopoietic stem and precursor cells in a leukopak can be increased by treating the donor with a cocktail of mobilization factors prior to leukapheresis. This induces the release of these precursors from the bone marrow into the blood, allowing them to be collected as part of the leukopak.
To further isolate PBMCs from leukopaks for even higher PBMC yield or to isolate T cells from leukopaks, consider Akadeum’s Buoyancy Activated Cell Sorting (BACS™) microbubble protocol for easy cell separation. This protocol leaves target cell concentrations untouched by the isolation protocol by employing negative selection techniques.
Peripheral whole blood samples contain blood plasma, which is mainly composed of water, hormones, proteins, lipids, and other contaminants that can confound research and analysis. Circulating blood also has many red blood cells, which are essential to the transport of oxygen throughout our bodies. In addition to red blood cells, platelets, and plasma, peripheral blood also contains PBMCs—roughly 2 million per milliliter.
The protocol for separating whole blood into individual cell populations is typically composed of cumbersome density gradient separation phases, which are necessary to deplete RBC and plasma content.
These separation steps can sometimes also damage the desired cells and thus impact the results of the experiment. Therefore, separating individual cell types from whole blood samples is generally difficult to scale.
To isolate PBMCs directly from a peripheral blood sample, it must first be diluted to allow for the clearest separation reaction. The density gradient is formed by the addition of Ficoll and centrifuging the sample for 30-40 minutes. This method forms four distinct layers that will need meticulous manual isolation via pipetting.
There are some instances where whole blood is preferable to a source of PBMCs. For example, in the study of neutrophils—white blood cells that function as our first line of immune defense—the fresh quality and high concentration of fragile neutrophils provided by whole blood are necessary for experimental success.
With so many complex approaches to isolating highly concentrated PBMC samples ready for downstream analysis, deciding on the right one for your experimental applications can be daunting. Akadeum offers gentle and easy solutions for every application and will save you time and resources within your lab.
The final yield of cells of interest is far too important to risk exposing to Ficoll or additional rounds of intense centrifugation and manual separation via pipette. Allow this revolutionary technology to become a part of your standard workflow so that PBMC isolation is successful every time.
Whether isolating PBMCs from whole blood, buffy coat, or leukopak, Akadeum’s BACS™ kits are unbeatable. Discover more about isolating PBMCs using Akadeum’s BACS Microbubble Technology.
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