EasySep™ Mouse Monocyte Enrichment Kit

Immunomagnetic Negative Selection Cell Isolation Kit

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Immunomagnetic Negative Selection Cell Isolation Kit
From: 598 CAD

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Overview

The EasySep™ Mouse Monocyte Enrichment Kit is designed to isolate monocytes from bone marrow cell suspensions or blood by negative selection. Unwanted cells are targeted for removal with biotinylated antibodies directed against non-monocytes and Tetrameric Antibody Complexes recognizing biotin and dextran-coated magnetic particles. Labeled cells are separated using an EasySep™ magnet without the use of columns. Desired cells are poured off into a new tube.
Advantages:

• Fast, easy-to-use and column-free
• Up to 98% purity (blood), 93% purity (bone marrow)
• Isolated cells are untouched
Components:
  • EasySep™ Mouse Monocyte Enrichment Kit (Catalog #19761)
    • EasySep™ Mouse Monocyte Enrichment Cocktail, 0.5 mL
    • EasySep™ Biotin Selection Cocktail, 1 mL
    • EasySep™ Magnetic Particles, 2 x 1 mL
    • Normal Rat Serum, 2 mL

  • RoboSep™ Mouse Monocyte Enrichment Kit with Filter Tips (Catalog #19761RF)
    • EasySep™ Mouse Monocyte Enrichment Cocktail, 0.5 mL
    • EasySep™ Biotin Selection Cocktail, 1 mL
    • EasySep™ Magnetic Particles, 2 x 1 mL
    • Normal Rat Serum, 2 mL
    • RoboSep™ Buffer (Catalog #20104)
    • RoboSep™ Filter Tips (Catalog #20125)
Magnet Compatibility:
• EasySep™ Magnet (Catalog #18000)
• “The Big Easy” EasySep™ Magnet (Catalog #18001)
• RoboSep™-S (Catalog #21000)
Subtype:
Cell Isolation Kits
Cell Type:
Monocytes
Species:
Mouse
Sample Source:
Bone Marrow; Whole Blood
Selection Method:
Negative
Application:
Cell Isolation
Brand:
EasySep; RoboSep
Area of Interest:
Immunology

Technical Resources

Educational Materials

(5)

Frequently Asked Questions

(16)

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.
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Data and Publications

Data

FACS Profile Results with EasySep™ Mouse Monocyte Enrichment Kit

Figure 1. FACS Profile Results with EasySep™ Mouse Monocyte Enrichment Kit

The CD11b+Ly-6G– cell content of the enriched cells typically ranges from 80% - 93% (Bone Marrow) and 92% - 98% (Blood). *Red blood cells were removed by lysis prior to flow cytometry.

Publications

(4)
Blood 2010 October

Lack of glucose recycling between endoplasmic reticulum and cytoplasm underlies cellular dysfunction in glucose-6-phosphatase-beta-deficient neutrophils in a congenital neutropenia syndrome.

Jun H et al.

Abstract

G6PC3 deficiency, characterized by neutropenia and neutrophil dysfunction, is caused by deficiencies in the endoplasmic reticulum (ER) enzyme glucose-6-phosphatase-β (G6Pase-β or G6PC3) that converts glucose-6-phosphate (G6P) into glucose, the primary energy source of neutrophils. Enhanced neutrophil ER stress and apoptosis underlie neutropenia in G6PC3 deficiency, but the exact functional role of G6Pase-β in neutrophils remains unknown. We hypothesized that the ER recycles G6Pase-β-generated glucose to the cytoplasm, thus regulating the amount of available cytoplasmic glucose/G6P in neutrophils. Accordingly, a G6Pase-β deficiency would impair glycolysis and hexose monophosphate shunt activities leading to reductions in lactate production, adenosine-5'-triphosphate (ATP) production, and reduced nicotinamide adenine dinucleotide phosphate (NADPH) oxidase activity. Using annexin V-depleted neutrophils, we show that glucose transporter-1 translocation is impaired in neutrophils from G6pc3(-/-) mice and G6PC3-deficient patients along with impaired glucose uptake in G6pc3(-/-) neutrophils. Moreover, levels of G6P, lactate, and ATP are markedly lower in murine and human G6PC3-deficient neutrophils, compared with their respective controls. In parallel, the expression of NADPH oxidase subunits and membrane translocation of p47(phox) are down-regulated in murine and human G6PC3-deficient neutrophils. The results establish that in nonapoptotic neutrophils, G6Pase-β is essential for normal energy homeostasis. A G6Pase-β deficiency prevents recycling of ER glucose to the cytoplasm, leading to neutrophil dysfunction.
Journal of leukocyte biology 2010 December

LPS-induced cytokine production in human dendritic cells is regulated by sialidase activity.

Stamatos N et al.

Abstract

Removal of sialic acid from glycoconjugates on the surface of monocytes enhances their response to bacterial LPS. We tested the hypothesis that endogenous sialidase activity creates a permissive state for LPS-induced cytokine production in human monocyte-derived DCs. Of the four genetically distinct sialidases (Neu1-4), Neu1, Neu3, and Neu4 are expressed in human monocytes, but only Neu1 and Neu3 are up-regulated as cells differentiate into DCs. Neu1 and Neu3 are present on the surface of monocytes and DCs and are also present intracellularly. DCs contain a greater amount of sialic acid than monocytes, but the amount of sialic acid/mg total protein declines during differentiation to DCs. This relative hyposialylation of cells does not occur in mature DCs grown in the presence of zanamivir, a pharmacologic inhibitor of Neu3 but not Neu1, or DANA, an inhibitor of Neu1 and Neu3. Inhibition of sialidase activity during differentiation to DCs causes no detectable change in cell viability or expression of DC surface markers. Differentiation of monocytes into DCs in the presence of zanamivir results in reduced LPS- induced expression of IL-6, IL-12p40, and TNF-α by mature DCs, demonstrating a role for Neu3 in cytokine production. A role for Neu3 is supported by inhibition of cytokine production by DANA in DCs from Neu1â�»/â�» and WT mice. We conclude that sialidase-mediated change in sialic acid content of specific cell surface glycoconjugates in DCs regulates LPS-induced cytokine production, thereby contributing to development of adaptive immune responses.
Journal of immunology (Baltimore, Md. : 1950) 2010 August

sRAGE induces human monocyte survival and differentiation.

Wang Y et al.

Abstract

The receptor for advanced glycation end products (RAGE) is produced either as a transmembrane or soluble form (sRAGE). Substantial evidence supports a role for RAGE and its ligands in disease. sRAGE is reported to be a competitive, negative regulator of membrane RAGE activation, inhibiting ligand binding. However, some reports indicate that sRAGE is associated with inflammatory disease. We sought to define the biological function of sRAGE on inflammatory cell recruitment, survival, and differentiation in vivo and in vitro. To test the in vivo impact of sRAGE, the recombinant protein was intratracheally administered to mice, which demonstrated monocyte- and neutrophil-mediated lung inflammation. We also observed that sRAGE induced human monocyte and neutrophil migration in vitro. Human monocytes treated with sRAGE produced proinflammatory cytokines and chemokines. Our data demonstrated that sRAGE directly bound human monocytes and monocyte-derived macrophages. Binding of sRAGE to monocytes promoted their survival and differentiation to macrophages. Furthermore, sRAGE binding to cells increased during maturation, which was similar in freshly isolated mouse monocytes compared with mature tissue macrophages. Because sRAGE activated cell survival and differentiation, we examined intracellular pathways that were activated by sRAGE. In primary human monocytes and macrophages, sRAGE treatment activated Akt, Erk, and NF-kappaB, and their activation appeared to be critical for cell survival and differentiation. Our data suggest a novel role for sRAGE in monocyte- and neutrophil-mediated inflammation and mononuclear phagocyte survival and differentiation.
Journal of molecular and cellular cardiology 2009 November

Dedifferentiated fat cells convert to cardiomyocyte phenotype and repair infarcted cardiac tissue in rats.

Jumabay M et al.

Abstract

Adipose tissue-derived stem cells have been demonstrated to differentiate into cardiomyocytes and vascular endothelial cells. Here we investigate whether mature adipocyte-derived dedifferentiated fat (DFAT) cells can differentiate to cardiomyocytes in vitro and in vivo by establishing DFAT cell lines via ceiling culture of mature adipocytes. DFAT cells were obtained by dedifferentiation of mature adipocytes from GFP-transgenic rats. We evaluated the differentiating ability of DFAT cells into cardiomyocytes by detection of the cardiac phenotype markers in immunocytochemical and RT-PCR analyses in vitro. We also examined effects of the transplantation of DFAT cells into the infarcted heart of rats on cardiomyocytes regeneration and angiogenesis. DFAT cells expressed cardiac phenotype markers when cocultured with cardiomyocytes and also when grown in MethoCult medium in the absence of cardiomyocytes, indicating that DFAT cells have the potential to differentiate to cardiomyocyte lineage. In a rat acute myocardial infarction model, transplanted DFAT cells were efficiently accumulated in infarcted myocardium and expressed cardiac sarcomeric actin at 8 weeks after the cell transplantation. The transplantation of DFAT cells significantly (p<0.05) increased capillary density in the infarcted area when compared with hearts from saline-injected control rats. We demonstrated that DFAT cells have the ability to differentiate to cardiomyocyte-like cells in vitro and in vivo. In addition, transplantation of DFAT cells led to neovascuralization in rats with myocardial infarction. We propose that DFAT cells represent a promising candidate cell source for cardiomyocyte regeneration in severe ischemic heart disease.
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