Easily and efficiently isolate highly purified human CD14+CD16+ monocytes from fresh or previously frozen human peripheral blood mononuclear cells (PBMCs) or lysed leukapheresis samples by immunomagnetic negative selection, with the EasySep™ Human Monocyte Enrichment Kit without CD16 Depletion. 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™ negative selection procedure, unwanted cells are labeled with antibody complexes and magnetic particles. The following unwanted cells are targeted for removal: granulocytes, T cells, B cells, subsets of dendritic cells, NK cells, and erythroid cells. The cocktail in this kit also contains an antibody to human Fc receptor to prevent non-specific binding. The magnetically labeled cells are then separated from the untouched desired monocytes by using an EasySep™ magnet and simply pouring or pipetting the desired cells into a new tube. Following magnetic cell isolation, the desired monocytes and CD14+CD16+ monocytes are ready for downstream applications such as flow cytometry, culture, or DNA/RNA extraction. The CD14+CD16+ subset of monocytes (~10% in blood of healthy individuals) has characteristics of tissue macrophages and expands greatly in acute and chronic inflammatory disease.
For applications in which removal of all CD16+ cells is desired, we recommend the EasySep™ Human Monocyte Enrichment Kit (Catalog #19359), which contains anti-CD16.
For large-scale isolation of CD14+CD16+/- and CD14-CD16+CD56- monocytes from lysed leukapheresis samples, see the large-format (1x10^10 cells) kit (Catalog #100-1525).
Learn more about how immunomagnetic EasySep™ technology works or how to fully automate immunomagnetic cell isolation with RoboSep™. Explore additional products optimized for your workflow, including culture media, supplements, antibodies, and more.
In this EasySep™ negative selection procedure, unwanted cells are labeled with antibody complexes and magnetic particles. The following unwanted cells are targeted for removal: granulocytes, T cells, B cells, subsets of dendritic cells, NK cells, and erythroid cells. The cocktail in this kit also contains an antibody to human Fc receptor to prevent non-specific binding. The magnetically labeled cells are then separated from the untouched desired monocytes by using an EasySep™ magnet and simply pouring or pipetting the desired cells into a new tube. Following magnetic cell isolation, the desired monocytes and CD14+CD16+ monocytes are ready for downstream applications such as flow cytometry, culture, or DNA/RNA extraction. The CD14+CD16+ subset of monocytes (~10% in blood of healthy individuals) has characteristics of tissue macrophages and expands greatly in acute and chronic inflammatory disease.
For applications in which removal of all CD16+ cells is desired, we recommend the EasySep™ Human Monocyte Enrichment Kit (Catalog #19359), which contains anti-CD16.
For large-scale isolation of CD14+CD16+/- and CD14-CD16+CD56- monocytes from lysed leukapheresis samples, see the large-format (1x10^10 cells) kit (Catalog #100-1525).
Learn more about how immunomagnetic EasySep™ technology works or how to fully automate immunomagnetic cell isolation with RoboSep™. Explore additional products optimized for your workflow, including culture media, supplements, antibodies, and more.
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Educational Materials (9)
Frequently Asked Questions
Publications (22)
Interleukin-4 receptor alpha signaling regulates monocyte homeostasis. FASEB journal : official publication of the Federation of American Societies for Experimental Biology 2022 oct
Abstract
Interleukin-4 (IL-4) and its receptors (IL-4R) promote the proliferation and polarization of macrophages. However, it is unknown if IL-4R also influences monocyte homeostasis and if steady state IL-4 levels are sufficient to affect monocytes. Employing full IL-4 receptor alpha knockout mice (IL-4R$\alpha$-/- ) and mice with a myeloid-specific deletion of IL-4R$\alpha$ (IL-4R$\alpha$f/f LysMcre ), we show that IL-4 acts as a homeostatic factor regulating circulating monocyte numbers. In the absence of IL-4R$\alpha$, murine monocytes in blood were reduced by 50% without altering monocytopoiesis in the bone marrow. This reduction was accompanied by a decrease in monocyte-derived inflammatory cytokines in the plasma. RNA sequencing analysis and immunohistochemical staining of splenic monocytes revealed changes in mRNA and protein levels of anti-apoptotic factors including BIRC6 in IL-4R$\alpha$-/- knockout animals. Furthermore, assessment of monocyte lifespan in vivo measuring BrdU+ cells revealed that the lifespan of circulating monocytes was reduced by 55% in IL-4R$\alpha$-/- mice, whereas subcutaneously applied IL-4 prolonged it by 75%. Treatment of human monocytes with IL-4 reduced the amount of dying monocytes in vitro. Furthermore, IL-4 stimulation reduced the phosphorylation of proteins involved in the apoptosis pathway, including the phosphorylation of the NF$\kappa$Bp65 protein. In a cohort of human patients, serum IL-4 levels were significantly associated with monocyte counts. In a sterile peritonitis model, reduced monocyte counts resulted in an attenuated recruitment of monocytes upon inflammatory stimulation in IL-4R$\alpha$f/f LysMcre mice without changes in overall migratory function. Thus, we identified a homeostatic role of IL-4R$\alpha$ in regulating the lifespan of monocytes in vivo.
BCG hydrogel promotes CTSS-mediated antigen processing and presentation, thereby suppressing metastasis and prolonging survival in melanoma. Journal for immunotherapy of cancer 2022 jun
Abstract
BACKGROUND The use of intralesional Mycobacterium bovis BCG (intralesional live BCG) for the treatment of metastatic melanoma resulted in regression of directly injected, and occasionally of distal lesions. However, intralesional-BCG is less effective in patients with visceral metastases and did not significantly improve overall survival. METHODS We generated a novel BCG lysate and developed it into a thermosensitive PLGA-PEG-PLGA hydrogel (BCG hydrogel), which was injected adjacent to the tumor to assess its antitumor effect in syngeneic tumor models (B16F10, MC38). The effect of BCG hydrogel treatment on contralateral tumors, lung metastases, and survival was assessed to evaluate systemic long-term efficacy. Gene expression profiles of tumor-infiltrating immune cells and of tumor-draining lymph nodes from BCG hydrogel-treated mice were analyzed by single-cell RNA sequencing (scRNA-seq) and CD8+ T cell receptor (TCR) repertoire diversity was assessed by TCR-sequencing. To confirm the mechanistic findings, RNA-seq data of biopsies obtained from in-transit cutaneous metastases of patients with melanoma who had received intralesional-BCG therapy were analyzed. RESULTS Here, we show that BCG lysate exhibits enhanced antitumor efficacy compared to live mycobacteria and promotes a proinflammatory tumor microenvironment and M1 macrophage (M$\Phi$) polarization in vivo. The underlying mechanisms of BCG lysate-mediated tumor immunity are dependent on M$\Phi$ and dendritic cells (DCs). BCG hydrogel treatment induced systemic immunity in melanoma-bearing mice with suppression of lung metastases and improved survival. Furthermore, BCG hydrogel promoted cathepsin S (CTSS) activity in M$\Phi$ and DCs, resulting in enhanced antigen processing and presentation of tumor-associated antigens. Finally, BCG hydrogel treatment was associated with increased frequencies of melanoma-reactive CD8+ T cells. In human patients with melanoma, intralesional-BCG treatment was associated with enhanced M1 M$\Phi$, mature DC, antigen processing and presentation, as well as with increased CTSS expression which positively correlated with patient survival. CONCLUSIONS These findings provide mechanistic insights as well as rationale for the clinical translation of BCG hydrogel as cancer immunotherapy to overcome the current limitations of immunotherapies for the treatment of patients with melanoma.
Activation of the Innate Immune Checkpoint CLEC5A on Myeloid Cells in the Absence of Danger Signals Modulates Macrophages' Function but Does Not Trigger the Adaptive T Cell Immune Response. Journal of immunology research 2022
Abstract
C-Type lectin receptor 5A (CLEC5A) is a spleen tyrosine kinase- (Syk-) coupled pattern recognition receptor expressed on myeloid cells and involved in the innate immune response to viral and bacterial infections. Activation of the CLEC5A receptor with pathogen-derived antigens leads to a secretion of proinflammatory mediators such as TNF-$\alpha$ and IL-6 that may provoke a systemic cytokine storm, and CLEC5A gene polymorphisms are associated with the severity of DV infection. In addition, the CLEC5A receptor was mentioned in the context of noninfectious disorders like chronic obstructive pulmonary disease (COPD) or arthritis. Altogether, CLEC5A may be considered as an innate immune checkpoint capable to amplify proinflammatory signals, and this way contributes to infection or to aseptic inflammation. In this study, we determined CLEC5A receptor expression on different macrophage subsets (in vitro and ex vivo) and the functional consequences of its activation in aseptic conditions. The CLEC5A surface expression appeared the highest on proinflammatory M1 macrophages while intermediate on tumor-associated phenotypes (M2c or TAM). In contrast, the CLEC5A expression on ex vivo-derived alveolar macrophages from healthy donors or macrophages from ovarian cancer patients was hardly detectable. Targeting CLEC5A on noninflammatory macrophages with an agonistic $\alpha$-CLEC5A antibody triggered a release of proinflammatory cytokines, resembling a response to dengue virus, and led to phenotypic changes in myeloid cells that may suggest their reprogramming towards a proinflammatory phenotype, e.g., upregulation of CD80 and downregulation of CD163. Interestingly, the CLEC5A agonist upregulated immune-regulatory molecules like CD206, PD-L1, and cytokines like IL-10, macrophage-derived chemokine (MDC/CCL22), and thymus and activation chemokine (TARC/CCL17) which are associated with an anti-inflammatory or a protumorigenic macrophage phenotype. In the absence of concomitant pathogenic or endogenous danger signals, the CLEC5A receptor activation did not amplify an autologous T cell response, which may represent a protective innate mechanism to avoid an undesirable autoimmune adaptive response.
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