EasySep™ Human Plasmacytoid DC 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™ Human Plasmacytoid DC Enrichment Kit

Immunomagnetic negative selection cell isolation kit

From: 1,109 USD
Catalog #
19062_C
Immunomagnetic negative selection cell isolation kit

Product Advantages


  • Fast, easy-to-use and column-free

  • Up to 97% purity

  • Untouched, viable cells

What's Included

  • EasySep™ Human Plasmacytoid DC Enrichment Kit (Catalog #19062)
    • EasySep™ Human Plasmacytoid DC Enrichment Cocktail Component A, 2 x 1 mL
    • EasySep™ Human DC Enrichment Cocktail Component B, 2 x 1 mL
    • EasySep™ D Magnetic Particles, 8 x 1 mL
    • Anti-Human CD32 (Fc gamma RII) Blocker, 2 x 0.8 mL
  • RoboSep™ Human Plasmacytoid DC Enrichment Kit with Filter Tips (Catalog #19062RF)
    • EasySep™ Human Plasmacytoid DC Enrichment Cocktail Component A, 2 x 1 mL
    • EasySep™ Human DC Enrichment Cocktail Component B, 2 x 1 mL
    • EasySep™ D Magnetic Particles, 8 x 1 mL
    • Anti-Human CD32 (Fc gamma RII) Blocker, 2 x 0.8 mL
    • RoboSep™ Buffer (Catalog #20104) x 2
    • RoboSep™ Filter Tips (Catalog #20125) x 2
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™ Human Plasmacytoid DC Enrichment Kit is designed to isolate plasmacytoid dendritic cells (pDC) from fresh peripheral blood mononuclear cells by negative selection. Unwanted cells are targeted for removal with Tetrameric Antibody Complexes recognizing non-pDCs and dextran-coated magnetic particles. The labeled cells are separated using the 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)
• EasyPlate™ EasySep™ Magnet (Catalog 18102)
• Easy 50 EasySep™ Magnet (Catalog #18002)
• RoboSep™-S (Catalog #21000)
Subtype
Cell Isolation Kits
Cell Type
Dendritic Cells
Species
Human
Sample Source
PBMC
Selection Method
Negative
Application
Cell Isolation
Brand
EasySep, RoboSep
Area of Interest
Immunology

Data Figures

Typical EasySep™ Human pDC Enrichment Profile

Figure 1. Typical EasySep™ Human pDC Enrichment Profile

Starting with 0.2 - 0.9% pDC in PBMC, the pDC content of the enriched fraction typically ranges from 87 - 97% purity based on the pDC phenotype of Lineage (CD3, CD14, CD16, CD19, CD20, CD34, CD56) negative, HLA-DR positive, and CD304 (BDCA-4) positive.

FACS Purity Data from pDC Enrichment Kit User

Figure 2. FACS Purity Data from pDC Kit User

FACS enrichment plots from Dr. Stuart R. McGregor Dallas of Princeton University. Prior to enrichment, the percentage of pDCs in total PBMC is approximately 0.1% (upper row), however following enrichment, a population of pDCs in excess of 97% pure can be obtained (lower row). pDCs identified using surface marker staining for CD303 and HLA-DR. Data originally posted as part of a product review in Biocompare.

Protocols and Documentation

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

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Safety Data Sheet 5
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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)

Differential and Overlapping Immune Programs Regulated by IRF3 and IRF5 in Plasmacytoid Dendritic Cells. K. T. Chow et al. Journal of immunology (Baltimore, Md. : 1950) 2018 NOV

Abstract

We examined the signaling pathways and cell type-specific responses of IFN regulatory factor (IRF) 5, an immune-regulatory transcription factor. We show that the protein kinases IKK$\alpha$, IKK$\beta$, IKK$\epsilon$, and TANK-binding kinase 1 each confer IRF5 phosphorylation/dimerization, thus extending the family of IRF5 activator kinases. Among primary human immune cell subsets, we found that IRF5 is most abundant in plasmacytoid dendritic cells (pDCs). Flow cytometric cell imaging revealed that IRF5 is specifically activated by endosomal TLR signaling. Comparative analyses revealed that IRF3 is activated in pDCs uniquely through RIG-I-like receptor (RLR) signaling. Transcriptomic analyses of pDCs show that the partitioning of TLR7/IRF5 and RLR/IRF3 pathways confers differential gene expression and immune cytokine production in pDCs, linking IRF5 with immune regulatory and proinflammatory gene expression. Thus, TLR7/IRF5 and RLR-IRF3 partitioning serves to polarize pDC response outcome. Strategies to differentially engage IRF signaling pathways should be considered in the design of immunotherapeutic approaches to modulate or polarize the immune response for specific outcome.
Diversification of human plasmacytoid predendritic cells in response to a single stimulus. Alculumbre SG et al. Nature immunology 2018 JAN

Abstract

Innate immune cells adjust to microbial and inflammatory stimuli through a process termed environmental plasticity, which links a given individual stimulus to a unique activated state. Here, we report that activation of human plasmacytoid predendritic cells (pDCs) with a single microbial or cytokine stimulus triggers cell diversification into three stable subpopulations (P1-P3). P1-pDCs (PD-L1+CD80-) displayed a plasmacytoid morphology and specialization for type I interferon production. P3-pDCs (PD-L1-CD80+) adopted a dendritic morphology and adaptive immune functions. P2-pDCs (PD-L1+CD80+) displayed both innate and adaptive functions. Each subpopulation expressed a specific coding- and long-noncoding-RNA signature and was stable after secondary stimulation. P1-pDCs were detected in samples from patients with lupus or psoriasis. pDC diversification was independent of cell divisions or preexisting heterogeneity within steady-state pDCs but was controlled by a TNF autocrine and/or paracrine communication loop. Our findings reveal a novel mechanism for diversity and division of labor in innate immune cells.
Enhanced plasmacytoid dendritic cell antiviral responses after omalizumab. Gill MA et al. The Journal of allergy and clinical immunology 2017 SEP

Abstract

BACKGROUND Atopy and viral respiratory tract infections synergistically promote asthma exacerbations. IgE cross-linking inhibits critical virus-induced IFN-α responses of plasmacytoid dendritic cells (pDCs), which can be deficient in patients with allergic asthma. OBJECTIVE We sought to determine whether reducing IgE levels in vivo with omalizumab treatment increases pDC antiviral IFN-α responses in inner-city children with asthma. METHODS PBMCs and pDCs isolated from children with exacerbation-prone asthma before and during omalizumab treatment were stimulated ex vivo with rhinovirus and influenza in the presence or absence of IgE cross-linking. IFN-α levels were measured in supernatants, and mRNA expression of IFN-α pathway genes was determined by using quantitative RT-PCR (qRT-PCR) in cell pellets. FcεRIα protein levels and mRNA expression were measured in unstimulated cells by using flow cytometry and qRT-PCR, respectively. Changes in these outcomes and associations with clinical outcomes were analyzed, and statistical modeling was used to identify risk factors for asthma exacerbations. RESULTS Omalizumab treatment increased rhinovirus- and influenza-induced PBMC and rhinovirus-induced pDC IFN-α responses in the presence of IgE cross-linking and reduced pDC surface FcεRIα expression. Omalizumab-induced reductions in pDC FcεRIα levels were significantly associated with a lower asthma exacerbation rate during the outcome period and correlated with increases in PBMC IFN-α responses. PBMC FcεRIα mRNA expression measured on study entry significantly improved an existing model of exacerbation prediction. CONCLUSIONS These findings indicate that omalizumab treatment augments pDC IFN-α responses and attenuates pDC FcεRIα protein expression and provide evidence that these effects are related. These results support a potential mechanism underlying clinical observations that allergic sensitization is associated with increased susceptibility to virus-induced asthma exacerbations.

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