EasySep™ Direct Human Neutrophil Isolation Kit

Immunomagnetic isolation of neutrophils directly from whole blood

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™ Direct Human Neutrophil Isolation Kit

Immunomagnetic isolation of neutrophils directly from whole blood

From: 545 USD
Catalog #
19666_C
Immunomagnetic isolation of neutrophils directly from whole blood

Product Advantages


  • > 99.9% RBC depletion without the need for density gradient centrifugation, sedimentation, or lysis

  • Up to 99% purity of isolated cells

  • Fast, easy-to-use and column-free

  • Isolated cells are untouched

What's Included

  • EasySep™ Direct Human Neutrophil Isolation Kit (Catalog #19666)
    • EasySep™ Direct Human Neutrophil Isolation Cocktail, 2 x 2.5 mL
    • EasySep™ Direct RapidSpheres™, 4 x 2.5 mL
  • RoboSep™ Human Neutrophil Isolation Kit (Catalog #100-0404)
    • EasySep™ Direct Human Neutrophil Isolation Cocktail, 2 x 2.5 mL
    • EasySep™ Direct RapidSpheres™, 4 x 2.5 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

What Our Scientist Says

RBC lysis and other preprocessing steps can complicate cell isolation procedures. We developed this kit so you can keep things simple and quickly isolate neutrophils directly from whole blood.

Christine BarrScientist
Christine Barr, Scientist

Overview

The EasySep™ Direct Human Neutrophil Isolation Kit isolates functional, highly purified neutrophils directly from human whole blood by immunomagnetic negative selection. No lysis, density gradient centrifugation or other processing steps are required and isolat​ed cells are immediately available for flow cytometry, functional assays, culture, and other downstream applications.

This product replaces the EasySep™ Human Neutrophil Isolation Kit (Catalog #19257) for even faster cell isolations.
Magnet Compatibility
• EasySep™ Magnet (Catalog #18000)
• “The Big Easy” EasySep™ Magnet (Catalog #18001)
• Easy 50 EasySep™ Magnet (Catalog #18002)
• EasyEights™ EasySep™ Magnet (Catalog #18103)
• RoboSep™-S (Catalog #21000)
Subtype
Cell Isolation Kits
Cell Type
Granulocytes and Subsets
Species
Human
Sample Source
Whole Blood
Selection Method
Negative
Application
Cell Isolation
Brand
EasySep, RoboSep
Area of Interest
Immunology, Infectious Diseases

Data Figures

Starting with Human Whole Blood from Normal Healthy Donors, the Typical Neutrophil (CD66b+CD16+) Content of the Non-lysed Final Isolated Fraction Is 97.3 ± 1.4% (Gated on CD45) or 94.0 ± 3.7% (Not Gated on CD45)

Figure 1. Typical EasySep™ Direct Human Neutrophil Isolation Profile

Starting with human whole blood from normal healthy donors, the typical neutrophil (CD66b+CD16+) content of the non-lysed final isolated fraction Is 97.3 ± 1.4% (gated on CD45) or 94.0 ± 3.7% (not gated on CD45). In the example above, the neutrophil (CD66b+CD16+) content of the lysed whole blood start sample and the non-lysed final isolated fraction is 50.6% and 97.2% (gated on CD45), respectively, or 43.6% and 95.9% (not gated on CD45), respectively. The starting frequency of neutrophils in the non-lysed whole blood start sample above is 0.04% (data not shown).

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 #
19666
Lot #
All
Language
English
Catalog #
100-0404
Lot #
All
Language
English
Document Type
Safety Data Sheet 1
Catalog #
19666
Lot #
All
Language
English
Document Type
Safety Data Sheet 2
Catalog #
19666
Lot #
All
Language
English
Document Type
Safety Data Sheet
Catalog #
100-0404
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 (12)

N-acetylcysteine inhibits thrombosis in a murine model of myeloproliferative neoplasm. B. M. Craver et al. Blood advances 2020 jan

Abstract

Thrombosis is a major cause of mortality in patients with myeloproliferative neoplasms (MPNs), though there is currently little to offer patients with MPN beyond aspirin and cytoreductive therapies such as hydroxyurea for primary prevention. Thrombogenesis in MPN involves multiple cellular mechanisms, including platelet activation and neutrophil-extracellular trap formation; therefore, an antithrombotic agent that targets one or more of these processes would be of therapeutic benefit in MPN. Here, we treated the JAK2V617F knockin mouse model of polycythemia vera with N-acetylcysteine (NAC), a sulfhydryl-containing compound with broad effects on glutathione replenishment, free radical scavenging, and reducing disulfide bonds, to investigate its antithrombotic effects in the context of MPN. Strikingly, NAC treatment extended the lifespan of JAK2V617F mice without impacting blood counts or splenomegaly. Using an acute pulmonary thrombosis model in vivo, we found that NAC reduced thrombus formation to a similar extent as the irreversible platelet inhibitor aspirin. In vitro analysis of platelet activation revealed that NAC reduced thrombin-induced platelet-leukocyte aggregate formation in JAK2V617F mice. Furthermore, NAC reduced neutrophil extracellular trap formation in primary human neutrophils from patients with MPN as well as healthy controls. These results provide evidence that N-acetylcysteine inhibits thrombosis in JAK2V617F mice and provide a pre-clinical rationale for investigating NAC as a therapeutic to reduce thrombotic risk in MPN.
The Regulatory Proteins Rtg1/3 Govern Sphingolipid Homeostasis in the Human-Associated Yeast Candida albicans. S. D. Moreno-Vel\'asquez et al. Cell reports 2020 jan

Abstract

Integrating nutrient sensing with the synthesis of complex molecules is a central feature of metabolism. Yet the regulatory mechanisms underlying such integration are often unknown. Here, we establish that the transcription regulators Rtg1/3 are key determinants of sphingolipid homeostasis in the human fungal pathogen Candida albicans. Quantitative analysis of the C. albicans lipidome reveals Rtg1/3-dependent alterations in all complex sphingolipids and their precursors, ceramides. Mutations in the regulators render the fungus susceptible to myriocin, a sphingolipid synthesis inhibitor. Rtg1/3 exert control on the expression of several enzymes involved in the synthesis of sphingolipids' building blocks, and the regulators are activated upon engulfment of C. albicans cells by human neutrophils. We demonstrate that Rtg1p and Rtg3p are regulated at two levels, one in response to sphingolipids and the other by the nutrient sensor TOR. Our findings, therefore, indicate that the Rtg1/3 system integrates nutrient sensing into the synthesis of complex lipids.
Neutrophil extracellular traps contribute to immunothrombosis in COVID-19 acute respiratory distress syndrome. E. A. Middleton et al. Blood 2020

Abstract

COVID-19 affects millions of patients worldwide, with clinical presentation ranging from isolated thrombosis to acute respiratory distress syndrome (ARDS) requiring ventilator support. Neutrophil extracellular traps (NETs) originate from decondensed chromatin released to immobilize pathogens, and they can trigger immunothrombosis. We studied the connection between NETs and COVID-19 severity and progression. We conducted a prospective cohort study of COVID-19 patients (n = 33) and age- and sex-matched controls (n = 17). We measured plasma myeloperoxidase (MPO)-DNA complexes (NETs), platelet factor 4, RANTES, and selected cytokines. Three COVID-19 lung autopsies were examined for NETs and platelet involvement. We assessed NET formation ex vivo in COVID-19 neutrophils and in healthy neutrophils incubated with COVID-19 plasma. We also tested the ability of neonatal NET-inhibitory factor (nNIF) to block NET formation induced by COVID-19 plasma. Plasma MPO-DNA complexes increased in COVID-19, with intubation (P {\textless} .0001) and death (P {\textless} .0005) as outcome. Illness severity correlated directly with plasma MPO-DNA complexes (P = .0360), whereas Pao2/fraction of inspired oxygen correlated inversely (P = .0340). Soluble and cellular factors triggering NETs were significantly increased in COVID-19, and pulmonary autopsies confirmed NET-containing microthrombi with neutrophil-platelet infiltration. Finally, COVID-19 neutrophils ex vivo displayed excessive NETs at baseline, and COVID-19 plasma triggered NET formation, which was blocked by nNIF. Thus, NETs triggering immunothrombosis may, in part, explain the prothrombotic clinical presentations in COVID-19, and NETs may represent targets for therapeutic intervention.

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