EasySep™ Mouse Neutrophil Enrichment Kit

Immunomagnetic negative selection cell isolation kit

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

EasySep™ Mouse Neutrophil Enrichment Kit

Immunomagnetic negative selection cell isolation kit

From: 732 USD
Catalog #
19762_C
Immunomagnetic negative selection cell isolation kit

Product Advantages


  • Fast, easy-to-use and column-free

  • Up to 93.7% (blood) and 88.7% (bone marrow) purity

  • Isolated cells are untouched

What's Included

  • 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)
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

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.
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

Data Figures

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.

Protocols and Documentation

Find supporting information and directions for use in the Product Information Sheet or explore additional protocols below.

Document Type
Product Name
Catalog #
Lot #
Language
Catalog #
19762
Lot #
All
Language
English
Catalog #
19762RF
Lot #
All
Language
English
Document Type
Safety Data Sheet 1
Catalog #
19762
Lot #
All
Language
English
Document Type
Safety Data Sheet 2
Catalog #
19762
Lot #
All
Language
English
Document Type
Safety Data Sheet 3
Catalog #
19762
Lot #
All
Language
English
Document Type
Safety Data Sheet 1
Catalog #
19762RF
Lot #
All
Language
English
Document Type
Safety Data Sheet 2
Catalog #
19762RF
Lot #
All
Language
English
Document Type
Safety Data Sheet 3
Catalog #
19762RF
Lot #
All
Language
English

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.

Resources and Publications

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.

Publications (19)

Macrophage Metabolism of Apoptotic Cell-Derived Arginine Promotes Continual Efferocytosis and Resolution of Injury. A. Yurdagul et al. Cell metabolism 2020 mar

Abstract

Continual efferocytic clearance of apoptotic cells (ACs) by macrophages prevents necrosis and promotes injury resolution. How continual efferocytosis is promoted is not clear. Here, we show that the process is optimized by linking the metabolism of engulfed cargo from initial efferocytic events to subsequent rounds. We found that continual efferocytosis is enhanced by the metabolism of AC-derived arginine and ornithine to putrescine by macrophage arginase 1 (Arg1) and ornithine decarboxylase (ODC). Putrescine augments HuR-mediated stabilization of the mRNA encoding the GTP-exchange factor Dbl, which activates actin-regulating Rac1 to facilitate subsequent rounds of AC internalization. Inhibition of any step along this pathway after first-AC uptake suppresses second-AC internalization, whereas putrescine addition rescues this defect. Mice lacking myeloid Arg1 or ODC have defects in efferocytosis in vivo and in atherosclerosis regression, while treatment with putrescine promotes atherosclerosis resolution. Thus, macrophage metabolism of AC-derived metabolites allows for optimal continual efferocytosis and resolution of injury.
NADPH oxidase controls pulmonary neutrophil infiltration in the response to fungal cell walls by limiting LTB4. Z. Song et al. Blood 2020 jan

Abstract

Leukocyte nicotinamide adenine dinucleotide phosphate (NADPH) oxidase plays a key role in host defense and immune regulation. Genetic defects in NADPH oxidase result in chronic granulomatous disease (CGD), characterized by recurrent bacterial and fungal infections and aberrant inflammation. Key drivers of hyper-inflammation induced by fungal cell walls in CGD are still incompletely defined. Here, we found that CGD (CYBB-null) neutrophils produced higher amounts of leukotriene B4 (LTB4) in vitro following activation with zymosan or Immune complexes, as compared to wild type (WT) neutrophils. This correlated with increased calcium influx in CGD neutrophils, which is restrained in WT neutrophils by the electrogenic activity of the NADPH oxidase. Increased LTB4 generation by CGD neutrophils was also augmented by paracrine cross-talk with the LTB4 receptor BLT1. CGD neutrophils formed more numerous and larger clusters in the presence of zymosan in vitro compared to WT, which was also LTB4- and BLT1-dependent. In zymosan-induced lung inflammation, focal neutrophil infiltrates were increased in CGD compared to WT mice and associated with higher LTB4 levels. Inhibiting LTB4 synthesis or antagonizing the BLT1 receptor following zymosan challenge reduced lung neutrophil recruitment in CGD to WT levels. Thus, LTB4 was the major driver of excessive neutrophilic lung inflammation in CGD mice in the early response to fungal cell walls, likely by a dysregulated feed-forward loop involving amplified neutrophil production of LTB4. This study identifies neutrophil LTB4 generation as a target of NADPH oxidase regulation, which could potentially be exploited therapeutically to reduce excessive inflammation in CGD.
Acute hyperketonaemia alters T-cell-related cytokine gene expression within stimulated peripheral blood mononuclear cells following prolonged exercise. D. M. Shaw et al. European journal of applied physiology 2020 jan

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.

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