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

(16)
European journal of applied physiology 2020 jan

Acute hyperketonaemia alters T-cell-related cytokine gene expression within stimulated peripheral blood mononuclear cells following prolonged exercise.

D. M. Shaw et al.

Abstract

PURPOSE We investigated the effect of the racemic $\beta$-hydroxybutyrate precursor, R,S-1,3-butanediol (BD), on T-cell-related cytokine gene expression within stimulated peripheral blood mononuclear cells (PBMC) following prolonged, strenuous exercise. METHODS A repeated-measures, randomised, crossover study was conducted in nine healthy, trained male cyclists (age, 26.7 ± 5.2 years; VO2peak, 63.9 ± 2.5 mL kg-1 min-1). Participants ingested 0.35 g kg-1 of BD or placebo 30 min before and 60 min during 85 min of steady-state (SS) exercise, which preceded a {\~{}} 30 min time-trial (TT) (7 kJ kg-1). Blood samples were collected at pre-supplement, pre-exercise, post-SS, post-TT and 1-h post-TT. Whole blood cultures were stimulated with Staphylococcal enterotoxin B (SEB) for 24 h to determine T-cell-related interleukin (IL)-4, IL-10 and interferon (IFN)-$\gamma$ mRNA expression within isolated PBMCs in vitro. RESULTS Serum cortisol, total circulating leukocyte and lymphocyte, and T-cell subset concentrations were similar between trials during exercise and recovery (all p {\textgreater} 0.05). BD ingestion increased T-cell-related IFN-$\gamma$ mRNA expression compared with placebo throughout exercise and recovery (p = 0.011); however, IL-4 and IL-10 mRNA expression and the IFN-$\gamma$/IL-4 mRNA expression ratio were unaltered (all p {\textgreater} 0.05). CONCLUSION Acute hyperketonaemia appears to transiently amplify the initiation of the pro-inflammatory T-cell-related IFN-$\gamma$ response to an immune challenge in vitro during and following prolonged, strenuous exercise; suggesting enhanced type-1 T-cell immunity at the gene level.
Molecular therapy : the journal of the American Society of Gene Therapy 2019 nov

Genetically Engineered Cell-Derived Nanoparticles for Targeted Breast Cancer Immunotherapy.

X. Shi et al.

Abstract

Exosomes are nanosized membranous vesicles secreted by a variety of cells. Due to their unique and pharmacologically important properties, cell-derived exosome nanoparticles have drawn significant interest for drug development. By genetically modifying exosomes with two distinct types of surface-displayed monoclonal antibodies, we have developed an exosome platform termed synthetic multivalent antibodies retargeted exosome (SMART-Exo) for controlling cellular immunity. Here, we apply this approach to human epidermal growth factor receptor 2 (HER2)-expressing breast cancer by engineering exosomes through genetic display of both anti-human CD3 and anti-human HER2 antibodies, resulting in SMART-Exos dually targeting T cell CD3 and breast cancer-associated HER2 receptors. By redirecting and activating cytotoxic T cells toward attacking HER2-expressing breast cancer cells, the designed SMART-Exos exhibited highly potent and specific anti-tumor activity both in vitro and in vivo. This work demonstrates preclinical feasibility of utilizing endogenous exosomes for targeted breast cancer immunotherapy and the SMART-Exos as a broadly applicable platform technology for the development of next-generation immuno-nanomedicines.
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.
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