Isolate highly purified human CD14+ cells from fresh or previously frozen human peripheral blood mononuclear cells (PBMCs) or washed leukapheresis samples by immunomagnetic positive selection, with the EasySep™ Human CD14 Positive Selection Kit II. Widely used in published research for more than 20 years, EasySep™ combines the specificity of monoclonal antibodies with the simplicity of a column-free magnetic system.
In this EasySep™ positive selection procedure, desired cells are labeled with antibody complexes recognizing CD14 and magnetic particles. Labeled cells are separated using an EasySep™ magnet and by simply pouring or pipetting off the unwanted cells. The cells of interest remain in the tube. Following magnetic cell isolation in as little as 22 minutes, the desired CD14+ cells are ready for downstream applications such as flow cytometry, culture, or DNA/RNA extraction. The CD14 antigen is strongly expressed on monocytes and macrophages and weakly on granulocytes. It is also expressed on most tissue macrophages.
This product replaces the EasySep™ Human CD14 Positive Selection Kit (Catalog #18058) for even faster cell isolations.
For large-scale isolation of human CD14+ cells from leukapheresis samples, see the large-format (1x10^10 cells) kit (Catalog #100-0694).
Learn more about how immunomagnetic EasySep™ technology works or how to fully automate immunomagnetic cell isolation with RoboSep™. Alternatively, choose ready-to-use, ethically sourced, primary Human Peripheral Blood CD14+ Monocytes, Frozen isolated with EasySep™ Human CD14 Positive Selection Kit II. Explore additional products optimized for your workflow, including culture media, supplements, antibodies, and more.
Figure 1. Typical EasySep™ Human CD14 Positive Selection II Isolation Profile
Starting with a single cell suspension of human PBMCs, the CD14+ cell content of the isolated fraction is typically 95.3 ± 4.5% (mean ± SD using the purple EasySep™ Magnet).
Figure 2. FACS Data for Anti-Human CD14 Antibody, Clone M5E2, Alexa Fluor® 488-Conjugated
(A) Flow cytometry analysis of human peripheral blood mononuclear cells (PBMCs) labeled with Anti-Human CD14 Antibody, Clone M5E2, Alexa Fluor® 488 (Catalog #60004AD) and Anti-Human CD45 Antibody, Clone HI30, APC (Catalog #60018AD). (B) Flow cytometry analysis of human PBMCs processed with the EasySep™ Human CD14 Positive Selection Kit (Catalog #17858) and labeled with Anti-Human CD14 Antibody, Clone M5E2, Alexa Fluor® 488. Histograms show labeling of PBMCs (Start) and isolated cells (Isolated). Labeling of start cells with Mouse IgG2a, kappa Isotype Control Antibody, Clone MOPC-173, Alexa Fluor® 488 (Catalog #60071AD) is shown (solid line histogram). (C) Flow cytometry analysis of human whole blood nucleated cells processed with the EasySep™ HLA Whole Blood CD33 Positive Selection Kit (Catalog #17885) and labeled with Anti-Human CD14 Antibody, Clone M5E2, Alexa Fluor® 488. Histograms show labeling of whole blood nucleated cells (Start) and isolated cells (Isolated). Labeling of start cells with Mouse IgG2a, kappa Isotype Control Antibody, Clone MOPC-173, Alexa Fluor® 488 is shown (solid line histogram).
Figure 3. FACS Data for Anti-Human CD14 Antibody, Clone M5E2, PE-Conjugated
(A) Flow cytometry analysis of human peripheral blood mononuclear cells (PBMCs) labeled with Anti-Human CD14 Antibody, Clone M5E2, PE (filled histogram; Catalog #60004PE), or Mouse IgG2a, kappa Isotype Control Antibody, Clone HI30, APC (Catalog #60018AZ). (B) Flow cytometry analysis of human PBMCs processed with the EasySep™ Human CD14 Positive Selection Kit (Catalog #17858) and labeled with Anti-Human CD14 Antibody, Clone M5E2, PE. Histograms show labeling of PBMCs (Start) and isolated cells (Isolated). Labeling of start cells with Mouse IgG2a, kappa Isotype Control Antibody, Clone MOPC-173, PE (Catalog #60071PE) is shown (solid line historgram). (C) Flow cytometry analysis of human whole blood nucleated cells processed with the EasySep™ HLA Whole Blood CD33 Positive Selection Kit (Catalog #17885) and labeled with Anti-Human CD14 Antibody, Clone M5E2, FITC. Histograms show labeling of whole blood nucleated cells (Start) and isolated cells (Isolated). Labeling of start cells with Mouse IgG2a, kappa Isotype Control Antibody, Clone MOPC-173, PE is shown (solid line histogram).
This product is designed for use in the following research area(s) as part of the highlighted workflow stage(s). Explore these workflows to learn more about the other products we offer to support each research area.
Can EasySep™ be used for either positive or negative selection?
Yes. The EasySep™ kits use either a negative selection approach by targeting and removing unwanted cells or a positive selection approach targeting desired cells. Depletion kits are also available for the removal of cells with a specific undesired marker (e.g. GlyA).
How does the separation work?
Magnetic particles are crosslinked to cells using Tetrameric Antibody Complexes (TAC). When placed in the EasySep™ Magnet, labeled cells migrate to the wall of the tube. The unlabeled cells are then poured off into a separate fraction.
Which columns do I use?
The EasySep™ procedure is column-free. That's right - no columns!
How can I analyze the purity of my enriched sample?
The Product Information Sheet provided with each EasySep™ kit contains detailed staining information.
Can EasySep™ separations be automated?
Yes. RoboSep™, the fully automated cell separator, automates all EasySep™ labeling and cell separation steps.
Can EasySep™ be used to isolate rare cells?
Yes. We recommend a cell concentration of 2x108 cells/mL and a minimum working volume of 100 µL. Samples containing 2x107 cells or fewer should be suspended in 100 µL of buffer.
Are the EasySep™ magnetic particles FACS-compatible?
Yes, the EasySep™ particles are flow cytometry-compatible, as they are very uniform in size and about 5000X smaller than other commercially available magnetic beads used with column-free systems.
Can the EasySep™ magnetic particles be removed after enrichment?
No, but due to the small size of these particles, they will not interfere with downstream applications.
Can I alter the separation time in the magnet?
Yes; however, this may impact the kit's performance. The provided EasySep™ protocols have already been optimized to balance purity, recovery and time spent on the isolation.
For positive selection, can I perform more than 3 separations to increase purity?
Yes, the purity of targeted cells will increase with additional rounds of separations; however, cell recovery will decrease.
How does the binding of the EasySep™ magnetic particle affect the cells? is the function of positively selected cells altered by the bound particles?
Hundreds of publications have used cells selected with EasySep™ positive selection kits for functional studies. Our in-house experiments also confirm that selected cells are not functionally altered by the EasySep™ magnetic particles.
If particle binding is a key concern, we offer two options for negative selection. The EasySep™ negative selection kits can isolate untouched cells with comparable purities, while RosetteSep™ can isolate untouched cells directly from whole blood without using particles or magnets.
Identification of Mechanisms by Which Genetic Susceptibility Loci Influence Systemic Sclerosis Risk Using Functional Genomics in Primary T Cells and Monocytes. D. Gonz\'alez-Serna et al. Arthritis & rheumatology (Hoboken, N.J.) 2023 jun
Abstract
OBJECTIVE Systemic sclerosis (SSc) is a complex autoimmune disease with a strong genetic component. However, most of the genes associated with the disease are still unknown because associated variants affect mostly noncoding intergenic elements of the genome. We used functional genomics to translate the genetic findings into a better understanding of the disease. METHODS Promoter capture Hi-C and RNA-sequencing experiments were performed in CD4+ T cells and CD14+ monocytes from 10 SSc patients and 5 healthy controls to link SSc-associated variants with their target genes, followed by differential expression and differential interaction analyses between cell types. RESULTS We linked SSc-associated loci to 39 new potential target genes and confirmed 7 previously known SSc-associated genes. We highlight novel causal genes, such as CXCR5, as the most probable candidate gene for the DDX6 locus. Some previously known SSc-associated genes, such as IRF8, STAT4, and CD247, showed cell type-specific interactions. We also identified 15 potential drug targets already in use in other similar immune-mediated diseases that could be repurposed for SSc treatment. Furthermore, we observed that interactions were directly correlated with the expression of important genes implicated in cell type-specific pathways and found evidence that chromatin conformation is associated with genotype. CONCLUSION Our study revealed potential causal genes for SSc-associated loci, some of them acting in a cell type-specific manner, suggesting novel biologic mechanisms that might mediate SSc pathogenesis.
Differentiation of circulating monocytes into macrophages with metabolically activated phenotype regulates inflammation in dyslipidemia patients. E. Berenice Mart\'inez-Shio et al. Clinical and experimental immunology 2022 may
Abstract
Macrophages are mediators of inflammation having an important role in the pathogenesis of cardiovascular diseases. Recently, a pro-inflammatory subpopulation, known as metabolically activated macrophages (MMe), has been described in conditions of obesity and metabolic syndrome where they are known to release cytokines that can promote insulin resistance. Dyslipidemia represents an important feature in metabolic syndrome and corresponds to one of the main modifiable risk factors for the development of cardiovascular diseases. Circulating monocytes can differentiate into macrophages under certain conditions. They correspond to a heterogeneous population, which include inflammatory and anti-inflammatory subsets; however, there is a wide spectrum of phenotypes. Therefore, we decided to investigate whether the metabolic activated monocyte (MoMe) subpopulation is already present under dyslipidemia conditions. Secondly, we assessed whether different levels of cholesterol and triglycerides play a role in the polarization towards the metabolic phenotype (MMe) of macrophages. Our results indicate that MoMe cells are found in both healthy and dyslipidemia patients, with cells displaying the following metabolic phenotype: CD14varCD36+ABCA1+PLIN2+. Furthermore, the percentages of CD14++CD68+CD80+ pro-inflammatory monocytes are higher in dyslipidemia than in healthy subjects. When analysing macrophage differentiation, we observed that MMe percentages were higher in the dyslipidemia group than in healthy subjects. These MMe have the ability to produce high levels of IL-6 and the anti-inflammatory cytokine IL-10. Furthermore, ABCA1 expression in MMe correlates with LDL serum levels. Our study highlights the dynamic contributions of metabolically activated macrophages in dyslipidemia, which may have a complex participation in low-grade inflammation due to their pro- and anti-inflammatory function.
Identification of cross-reactive CD8+ T cell receptors with high functional avidity to a SARS-CoV-2 immunodominant epitope and its natural mutant variants. C. Hu et al. Genes & diseases 2022 jan
Abstract
Despite the growing knowledge of T cell responses in COVID-19 patients, there is a lack of detailed characterizations for T cell-antigen interactions and T cell functions. Here, with a predicted peptide library from SARS-CoV-2 S and N proteins, we found that specific CD8+ T cell responses were identified in over 75% of COVID-19 convalescent patients (15/20) and an epitope from the N protein, N361-369 (KTFPPTEPK), was the most dominant epitope from our selected peptide library. Importantly, we discovered 2 N361-369-specific T cell receptors (TCRs) with high functional avidity that were independent of the CD8 co-receptor. These TCRs exhibited complementary cross-reactivity to several presently reported N361-369 mutant variants, as to the wild-type epitope. Further, the natural functions of these TCRs in the cytotoxic immunity against SARS-CoV-2 were determined with dendritic cells (DCs) and the lung organoid model. We found that the N361-369 epitope could be normally processed and endogenously presented by these different types of antigen presenting cells, to elicit successful activation and effective cytotoxicity of CD8+ T cells ex vivo. Our study evidenced potential mechanisms of cellular immunity to SARS-CoV-2, and illuminated potential ways of viral clearance in COVID-19 patients. These results indicate that utilizing CD8-independent TCRs against SARS-CoV-2-associated antigens may provide functional superiority that is beneficial for the adoptive cell immunotherapies based on natural or genetically engineered T cells. Additionally, this information is highly relevant for the development of the next-generation vaccines with protections against continuously emerged SARS-CoV-2 mutant strains.
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