EasySep™ Human Neutrophil Enrichment Kit

Immunomagnetic Negative Selection Cell Isolation Kit

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Immunomagnetic Negative Selection Cell Isolation Kit
From: 936 CAD

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Overview

The EasySep™ Human Neutrophil Enrichment Kit is designed to isolate neutrophils from a polymorphonuclear cell-rich fraction of peripheral blood by negative selection. Unwanted cells are targeted for removal with Tetrameric Antibody Complexes recognizing non-neutrophils and magnetic particles. Labeled cells are separated using an EasySep™ magnet without the use of columns. Desired cells are poured off into a new tube. The enrichment kit is compatible with cells prepared using HetaSep™ (Catalog #07906) sedimentation or red blood cell lysis.
Advantages:
• Fast, easy-to-use and column-free
• Up to 99% purity
• Untouched, viable cells
Components:
  • EasySep™ Human Neutrophil Enrichment Kit (Catalog #19257)
    • EasySep™ Human Neutrophil Enrichment Cocktail, 1 mL
    • EasySep™ Magnetic Particles, 3 x 1 mL
  • RoboSep™ Human Neutrophil Enrichment Kit with Filter Tips (Catalog #19257RF)
    • EasySep™ Human Neutrophil Enrichment Cocktail, 1 mL
    • EasySep™ Magnetic Particles, 3 x 1 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:
Granulocytes and Subsets
Species:
Human
Sample Source:
PMNC; Whole Blood
Selection Method:
Negative
Application:
Cell Isolation
Brand:
EasySep; RoboSep
Area of Interest:
Immunology

Technical Resources

Educational Materials

(6)

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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Product Applications

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.

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Data and Publications

Data

FACS Profile Results Using EasySep™ Human Neutrophil Enrichment Kit

Figure 1. FACS Profile Results Using EasySep™ Human Neutrophil Enrichment Kit

Starting with fresh HetaSep™-treated blood, the neutrophil content of the enriched fraction typically ranges from 98% - 99%.

Publications

(6)
Transplantation Direct 9000 December

Characterizing the Mechanistic Pathways of the Instant Blood-Mediated Inflammatory Reaction in Xenogeneic Neonatal Islet Cell Transplantation.

Liuwantara D et al.

Abstract

Introduction: The instant blood-mediated inflammatory reaction (IBMIR) causes major loss of islets after transplantation and consequently represents the initial barrier to survival of porcine neonatal islet cell clusters (NICC) after xenotransplantation. Methods: This study used novel assays designed to characterize the various immunologic components responsible for xenogeneic IBMIR to identify initiators and investigate processes of IBMIR-associated coagulation, complement activation and neutrophil infiltration. The IBMIR was induced in vitro by exposing NICC to platelet-poor or platelet-rich human plasma or isolated neutrophils. Results: We found that xenogeneic IBMIR was characterized by rapid, platelet-independent thrombin generation, with addition of platelets both accelerating and exacerbating this response. Platelet-independent complement activation was observed as early as 30 minutes after NICC exposure to plasma. However, membrane attack complex formation was not observed in NICC histopathology sections until after 60 minutes. We demonstrated for the first time that NICC-mediated complement activation was necessary for neutrophil activation in the xenogeneic IBMIR setting. Finally, using the Seahorse extracellular flux analyzer, we identified substantial loss of islet function (up to 40%) after IBMIR with surviving NICC showing evidence of mitochondrial damage. Conclusions: This study used novel assays to describe multiple key pathways by which xenogeneic IBMIR causes islet destruction, allowing further refinement of future interventions aimed at resolving the issue of IBMIR in xenotransplantation. This is an open-access article distributed under the terms of the Creative Commons Attribution-Non Commercial-No Derivatives License 4.0 (CCBY-NC-ND), where it is permissible to download and share the work provided it is properly cited. The work cannot be changed in any way or used commercially. (C) 2016 The Authors. Published by Wolters Kluwer Health, Inc.
American journal of respiratory cell and molecular biology 2011 June

Granulocyte/macrophage colony-stimulating factor causes a paradoxical increase in the BH3-only pro-apoptotic protein Bim in human neutrophils.

Cowburn A et al.

Abstract

Neutrophil apoptosis is essential for the resolution of inflammation but is delayed by several inflammatory mediators. In such terminally differentiated cells it has been uncertain whether these agents can inhibit apoptosis through transcriptional regulation of anti-death (Bcl-X(L), Mcl-1, Bcl2A1) or BH3-only (Bim, Bid, Puma) Bcl2-family proteins. We report that granulocyte/macrophage colony-stimulating factor (GM-CSF) and tumor necrosis factor (TNF)-α prevent the normal time-dependent loss of Mcl-1 and Bcl2A1 in neutrophils, and we demonstrate that they cause an NF-κB-dependent increase in Bcl-X(L) transcription/translation. We show that GM-CSF and TNF-α increase and/or maintain mRNA levels for the pro-apoptotic BH3-only protein Bid and that GM-CSF has a similar NF-κB-dependent effect on Bim transcription and BimEL expression. The in-vivo relevance of these findings was indicated by demonstrating that GM-CSF is the dominant neutrophil survival factor in lung lavage from patients with ventilator-associated pneumonia, confirming an increase in lung neutrophil Bim mRNA. Finally GM-CSF caused mitochondrial location of Bim and a switch in phenotype to a cell that displays accelerated caspase-9-dependent apoptosis. This study demonstrates the capacity of neutrophil survival agents to induce a paradoxical increase in the pro-apoptotic proteins Bid and Bim and suggests that this may function to facilitate rapid apoptosis at the termination of the inflammatory cycle.
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.
International immunology 2010 October

Neutrophil activation and survival are modulated by interaction with NK cells.

Costantini C et al.

Abstract

It is increasingly evident that neutrophils are able to cross-talk with other leukocytes to shape ongoing inflammatory and immune responses. In this study, we analyzed whether human NK cells may influence the survival and activation of neutrophils under co-culture conditions. We report that NK cells exposed to either IL-15 or IL-18 alone strongly protect the survival of neutrophils via the release of IFNγ and granulocyte macrophage colony-stimulating factor (GM-CSF) plus IFNγ, respectively, and cause a slight up-regulation of neutrophil CD64 and CD11b expression. In comparison, NK cells exposed to both IL-15 and IL-18 show a lesser ability to increase the survival of neutrophils but can more potently up-regulate CD64 and CD11b expression, as well as induce the de novo surface expression of CD69, in neutrophils. Analysis of the events occurring in neutrophil/NK co-cultures exposed to IL-15 plus IL-18 revealed that (i) neutrophil survival is positively affected by NK-derived GM-CSF but negatively influenced by a CD18-dependent neutrophil/NK contact, (ii) NK-derived IFNγ is almost entirely responsible for the induction of CD64, (iii) both soluble factors (primarily GM-CSF) and direct cell-cell contact up-regulate CD11b and CD69 and (iv) NK-derived GM-CSF induces the expression of biologically active heparin-binding EGF-like growth factor (HB-EGF) in neutrophils. Finally, we demonstrate that NK cells can also express HB-EGF when stimulated with either IL-2 or IL-15, yet independently of endogenous GM-CSF. Altogether, our results define a novel interaction within the innate immune system whereby NK cells, by directly modulating neutrophil functions, might contribute to the pathogenesis of inflammatory diseases.
Thorax 2010 December

Interleukin-5 inhibits glucocorticoid-mediated apoptosis in human eosinophils.

Brode S et al.

Abstract

Abstract not available.
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