Adaptive Biotechnologies is harnessing this vast system of biology to unleash its power as a natural diagnostic and therapeutic tool to propel a paradigm shift in medicine.
Isolating monocytes requires blood. Lots and lots of blood, depending on your downstream assay of choice. When planning your experiments, it is important to remember that monocytes have a half-life of days. Ensure you have signed any relevant COSHH forms and follow all safety procedures when handling human samples.
PBMCs are any blood cells that have a round nucleus. One quick and easy way to isolate monocytes from PBMCs is to use immunomagnetic negative selection cell separation. Immunomagnetic positive selection involves targeting the cells you are interested in with antibody complexes that recognise cell surface antigens specific to that cell type; e.
There are several kits out there that perform this task very efficiently; however, they can be expensive. The magnet usually must be purchased separately.
I can personally vouch for the negative selection Stemcell kit; however, there are others out there that are likely just as effective. Miltenyi Biotec and ThermoFisher also manufacture similar products. As the process uses a kit, it is straightforward and quick.
These isolated monocytes should be used immediately; as mentioned earlier, they have a half-life of days so you want them to be as fresh as possible. They are suitable for activation with cytokines, incubation with compounds, migration assays, flow cytometry, and many more assays. HSCs give rise to all blood cells of the immune system through a process called hematopoiesis.
As hematopoietic stem cells progress through hematopoiesis they generate the myeloid monocytes, macrophages, granulocytes, megakaryocytes, dendritic cells, erythrocytes and lymphoid T cells, B cells, NK cells lineages Figure 1. Within both lineages are cells that make up the PBMCs. PBMCs are blood cells with round nuclei that encompass a heterogeneous cell population comprising various frequencies of lymphocytes T cells, B cells, and NK cells , dendritic cells, and monocytes Table 1.
These cells are critical components of the innate and adaptive immune system which defends the body against viral, bacterial, and parasitic infection and destroys tumor cells and foreign substances. Lymphocytes play an essential role in cell-mediated and humoral immune responses, primarily associated with the activation of T and B cells. Once active, T cells launch a cell-mediated immune response that targets antigens within an infected or diseased cell.
Once activated, B cells differentiate into plasma cells capable of secreting antibodies that specifically target free antigens circulating in the bloodstream. The ability to target free antigens by secreted antibodies within the extracellular space is defined as the humoral immune response. Table 1. Average cell frequencies from enriched PBMC fractions. These cells perform their effector function without requiring an antigen and defend the body against tumor activity.
Dendritic cells, a highly specialized antigen-presenting cell, engulf antigens and present fragments of the antigen to cells of the adaptive immune system eliciting activation of T and B cells. Last, but of great importance, are the hematopoietic stem cells. HSCs within the blood and bone marrow give rise to all the cells within the blood, including red blood cells, platelets, lymphocytes, monocytes, and granulocytes.
Although desirable for stem cell transplants, this rare cell population accounts for only 0. Two primary techniques that separate peripheral blood mononuclear cells from whole peripheral blood are through the use of a density gradient centrifugation process or by leukapheresis.
Since cells have a specific density, the use of a density gradient centrifugation process separates the main cell populations, such as lymphocytes, monocytes, granulocytes, and red blood cells, throughout a density gradient medium.
For human cells, the medium will have a density of 1. After centrifugation, the PBMC fraction will appear as a thin white layer at the interface between the plasma and the density gradient medium making it easy to remove the PBMC fraction. Because of these differences in the phenotype of cells present in blood and in lamina propria of the gastrointestinal tract it can be expected that mononuclear cells isolated from blood will respond differently from lamina propria mononuclear cells.
Care should therefore be taken in drawing conclusions about the mucosal immune response in the intestine from in vitro studies with PBMCs. Blood is diluted with 2—4 volumes phosphate-buffered saline PBS pH The more diluted blood samples the better purity of the mononuclear cells. Add 35 ml of diluted blood to a 50 ml tube Fig. Using a brake during centrifugation will disturb the separation of the upper and lower fractions.
Aspirate most of the upper layer leaving the mononuclear cells at the interphase Fig. This step will remove platelets, and it should be repeated at least once.
Cells can be used immediately or preserved and stored in liquid nitrogen. Dilute the test compounds in RPMI with additions as described above. An array of methods and kit-based assay systems are available for this purpose, for example incorporation of 3 H-thymidine or measurement of mitochondrial reductase with tetrazolium salts MTT.
The optimal concentrations of the polyclonal activators can be established by cultivating PBMCs in the presence of twofold dilutions of the activators. The vehicle control solvent of the test compound stock should be included too. As background control, RPMI with the test compound and activators can be used. Upon stimulation with polyclonal activators PBMCs will produce cytokines and up-regulate activation markers.
Characterization of the cytokine profile and changes in activation marker expression, especially on T cells, may provide information whether the response is in the direction of Th1, Th2, Th17 or regulatory T cells. Investigations of the cytokine profile is most easily carried out by ELISA analysis of secreted cytokines in the culture supernatant. Alternatively, the number of cells producing the different cytokines can be studied after intracellular staining and analysis by flow cytometry.
Also, changes in gene expression levels by qPCR can be informative. Test compounds should be diluted in RPMI In accordance with literature [ 26 ], lipopolysaccharide from E.
Thus, isolated blood cells show higher sensitivity to stimulation with lipopolysaccharide as compared to whole blood, which may be due to interaction of LPS with other blood components, such as lipoproteins. These findings support the concept of selective mediator modulation as supportive therapy for sepsis rather than the specific targeting of individual factors.
The excellent technical support by Ingrid Linsberger is gratefully acknowledged. This is an open access article distributed under the Creative Commons Attribution License , which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
Article of the Year Award: Outstanding research contributions of , as selected by our Chief Editors. Read the winning articles. Journal overview. Special Issues. Received 08 Feb Revised 23 Mar Accepted 25 Mar Published 29 May Abstract THP-1 cells are widely applied to mimic monocytes in cell culture models. Introduction As a barrier between the blood stream and the surrounding tissues, the endothelium is involved in regulation of blood flow, vascular tone, thrombosis, thrombolysis, adherence of platelets, and extravasation of circulating leukocytes [ 1 , 2 ].
Materials and Methods 2. Blood and Plasma Blood was freshly drawn from healthy volunteers after informed written consent and anticoagulated with sodium citrate at a final concentration of Figure 1. Scheme of the cell culture model. The harvested conditioned medium containing LPS and cytokines is treated with adsorbents to modulate mediators of inflammation and is subsequently used to stimulate human umbilical vein endothelial cells HUVEC.
Figure 2. Unstimulated cultures served as control white circles. Figure 3. Black bars indicate control media without LPS. Figure 4. Effect of LPS on cytokine secretion from whole blood. Figure 5. Figure 6. Effect of mediator modulation on adhesion molecule expression. The experimental setup was identical to Figure 5. References W. Pober and W. Hack, S. Zeerleder, J. Dhainaut, F. Taylor, and K.
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