EasySep™ Mouse Neutrophil Enrichment Kit

Immunomagnetic negative selection cell isolation kit

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Immunomagnetic negative selection cell isolation kit
From: 628 USD

New look, same high quality and support! You may notice that your instrument or reagent packaging looks slightly different from images displayed on the website, or from previous orders. We are updating our look but rest assured, the products themselves and how you should use them have not changed. Learn more

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Overview

The EasySep™ Mouse Neutrophil Enrichment Kit is designed to isolate neutrophils from single-cell suspensions of bone marrow or other tissues by negative selection. Unwanted cells are targeted for removal with biotinylated antibodies that are directed against non-neutrophils. Labeled cells are then recognized by Tetrameric Antibody Complexes that are directed against biotin and dextran. These cells are bound to magnetic particles and 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 93.7% (blood) and 88.7% (bone marrow) purity
• Isolated cells are untouched
Components:
  • EasySep™ Mouse Neutrophil Enrichment Kit (Catalog #19762)
    • EasySep™ Mouse Neutrophil 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 Neutrophil Enrichment Kit with Filter Tips (Catalog #19762RF)
    • EasySep™ Mouse Neutrophil 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:
Granulocytes and Subsets
Species:
Mouse
Sample Source:
Bone Marrow; Whole Blood
Selection Method:
Negative
Application:
Cell Isolation
Brand:
EasySep; RoboSep
Area of Interest:
Immunology

Scientific Resources

Educational Materials

(5)

Frequently Asked Questions

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.

Research Area Workflow Stages for
Workflow Stages

Data and Publications

Data

EasySep™ Mouse Neutrophil Kit Isolation Profile

Figure 1. Typical FACS Profiles for EasySep™ Mouse Neutrophil Kit

The CD11b+Ly6G+ cell content of the enriched cells typically ranges from 69.9% - 88.7% with bone marrow samples, and 80.1% - 93.7% with blood samples.

Publications

(14)
Nature immunology 2019 feb

A noncanonical role for the engulfment gene ELMO1 in neutrophils that promotes inflammatory arthritis.

S. Arandjelovic et al.

Abstract

Rheumatoid arthritis is characterized by progressive joint inflammation and affects {\~{}}1{\%} of the human population. We noted single-nucleotide polymorphisms (SNPs) in the apoptotic cell-engulfment genes ELMO1, DOCK2, and RAC1 linked to rheumatoid arthritis. As ELMO1 promotes cytoskeletal reorganization during engulfment, we hypothesized that ELMO1 loss would worsen inflammatory arthritis. Surprisingly, Elmo1-deficient mice showed reduced joint inflammation in acute and chronic arthritis models. Genetic and cell-biology studies revealed that ELMO1 associates with receptors linked to neutrophil function in arthritis and regulates activation and early neutrophil recruitment to the joints, without general inhibition of inflammatory responses. Further, neutrophils from the peripheral blood of human donors that carry the SNP in ELMO1 associated with arthritis display increased migratory capacity, whereas ELMO1 knockdown reduces human neutrophil migration to chemokines linked to arthritis. These data identify 'noncanonical' roles for ELMO1 as an important cytoplasmic regulator of specific neutrophil receptors and promoter of arthritis.
Scientific reports 2019 apr

Cold-inducible RNA-binding Protein Induces Neutrophil Extracellular Traps in the Lungs during Sepsis.

Y. Ode et al.

Abstract

Extracellular cold-inducible RNA-binding protein (CIRP) exaggerates inflammation and tissue injury in sepsis. Neutrophil extracellular traps (NETs) are released by activated neutrophils during sepsis. NETs contribute to pathogen clearance, but excessive NET formation (NETosis) causes inflammation and tissue damage. Peptidylarginine deiminase 4 (PAD4) is associated with NETosis by increasing histone citrullination and chromatin decondensation. We hypothesized that CIRP induces NETosis in the lungs during sepsis via upregulating PAD4 expression. Sepsis was induced in C57BL/6 wild-type (WT) and CIRP-/- mice by cecal ligation and puncture (CLP). After 20 h of CLP induction, NETs in the lungs of WT and CIRP-/- mice were quantified by flow cytometry by staining the single cell suspensions with MPO and CitH3 Abs. PAD4 expression in the lungs of WT and CIRP-/- mice after sepsis was assessed by Western blotting. In vitro effects of recombinant mouse (rm) CIRP for NETosis and PAD4 expression in the bone marrow-derived neutrophils (BMDN) were assessed by flow cytometry and Western blotting, respectively. After 20 h of CLP, NETosis in the lungs was significantly decreased in CIRP-/- mice compared to WT mice, which also correlated with the decreased PAD4 expression. Intratracheal administration of rmCIRP into WT mice significantly increased NETosis and PAD4 expression in the lungs compared to vehicle-injected mice. In vitro culture of BMDN with rmCIRP significantly increased NETosis and PAD4 expression compared to PBS-treated control. Fluorescence microscopy revealed typical web-like structures consistent with NETs in rmCIRP-treated BMDN. Thus, CIRP serves as a novel inducer of NETosis via PAD4 during sepsis.
JCI insight 2019 apr

Deficiency of Socs3 leads to brain-targeted EAE via enhanced neutrophil activation and ROS production.

Z. Yan et al.

Abstract

Dysregulation of the JAK/STAT signaling pathway is associated with Multiple Sclerosis (MS) and its mouse model, Experimental Autoimmune Encephalomyelitis (EAE). Suppressors Of Cytokine Signaling (SOCS) negatively regulate the JAK/STAT pathway. We previously reported a severe, brain-targeted, atypical form of EAE in mice lacking Socs3 in myeloid cells (Socs3DeltaLysM), which is associated with cerebellar neutrophil infiltration. There is emerging evidence that neutrophils are detrimental in the pathology of MS/EAE, however, their exact function is unclear. Here we demonstrate that neutrophils from the cerebellum of Socs3DeltaLysM mice show a hyper-activated phenotype with excessive production of reactive oxygen species (ROS) at the peak of EAE. Neutralization of ROS in vivo delayed the onset and reduced severity of atypical EAE. Mechanistically, Socs3-deficient neutrophils exhibit enhanced STAT3 activation, a hyper-activated phenotype in response to G-CSF, and upon G-CSF priming, increased ROS production. Neutralization of G-CSF in vivo significantly reduced the incidence and severity of the atypical EAE phenotype. Overall, our work elucidates that hypersensitivity of G-CSF/STAT3 signaling in Socs3DeltaLysM mice leads to atypical EAE by enhanced neutrophil activation and increased oxidative stress, which may explain the detrimental role of G-CSF in MS patients.
JCI insight 2019 apr

Aged marrow macrophages expand platelet-biased hematopoietic stem cells via Interleukin1B.

B. J. Frisch et al.

Abstract

The bone marrow microenvironment (BMME) contributes to the regulation of hematopoietic stem cell (HSC) function, though its role in age-associated lineage skewing is poorly understood. Here we show that dysfunction of aged marrow macrophages (Mphis) directs HSC platelet-bias. Mphis from the marrow of aged mice and humans exhibited an activated phenotype, with increased expression of inflammatory signals. Aged marrow Mphis also displayed decreased phagocytic function. Senescent neutrophils, typically cleared by marrow Mphis, were markedly increased in aged mice, consistent with functional defects in Mphi phagocytosis and efferocytosis. In aged mice, Interleukin 1B (IL1B) was elevated in the bone marrow and caspase 1 activity, which can process pro-IL1B, was increased in marrow Mphis and neutrophils. Mechanistically, IL1B signaling was necessary and sufficient to induce a platelet bias in HSCs. In young mice, depletion of phagocytic cell populations or loss of the efferocytic receptor Axl expanded platelet-biased HSCs. Our data support a model wherein increased inflammatory signals and decreased phagocytic function of aged marrow Mphis induce the acquisition of platelet bias in aged HSCs. This work highlights the instructive role of Mphis and IL1B in the age-associated lineage-skewing of HSCs, and reveals the therapeutic potential of their manipulation as antigeronic targets.
Cell host microbe 2019 apr

Cleaved Cochlin Sequesters Pseudomonas aeruginosa and Activates Innate Immunity in the Inner Ear.

J. Jung et al.

Abstract

In the inner ear, endolymph fluid surrounds the organ of Corti, which is important for auditory function; notably, even slight environmental changes mediated by trauma or infection can have significant consequences. However, it is unclear how the immune response is modulated in these tissues. Here, we report the local immune surveillance role of cleaved cochlin LCCL (Limulus factor C, Cochlin, and Lgl1) during Pseudomonas aeruginosa infection in the cochlea. Upon infection, the LCCL domain is cleaved from cochlin and secreted into the perilymph. This cleaved fragment sequesters infiltrating bacteria in the scala tympani and subsequently recruits resident immune cells to eliminate the bacteria. Importantly, hearing loss in a cochlin knockout mouse model is remedied by treatment with a cochlin LCCL peptide. These findings suggest cleaved cochlin LCCL constitutes a critical factor in innate immunity and auditory function and may be a potential therapeutic target to treat chronic otitis media-induced hearing loss.
STEMCELL TECHNOLOGIES INC.’S QUALITY MANAGEMENT SYSTEM IS CERTIFIED TO ISO 13485. PRODUCTS ARE FOR RESEARCH USE ONLY AND NOT INTENDED FOR HUMAN OR ANIMAL DIAGNOSTIC OR THERAPEUTIC USES UNLESS OTHERWISE STATED.