EasySep™ Human Myeloid DC Enrichment Kit

Immunomagnetic negative selection cell isolation kit

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From: 798 USD

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Immunomagnetic negative selection cell isolation kit
From: 798 USD

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Overview

The EasySep™ Human Myeloid DC Enrichment Kit is designed to isolate myeloid dendritic cells (mDC) from fresh peripheral blood mononuclear cells or ammonium chloride-lysed leukapheresis by negative selection. Unwanted cells are targeted for removal with Tetrameric Antibody Complexes recognizing non-mDCs 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.
Advantages:
• Fast, easy-to-use and column-free
• Up to 90% purity
• Untouched, viable cells
Components:
  • EasySep™ Human Myeloid DC Enrichment Kit (Catalog #19061)
    • EasySep™ Human Myeloid DC Enrichment Cocktail Component A, 2 x 1 mL
    • EasySep™ Human DC Enrichment Cocktail Component B, 2 x 1 mL
    • EasySep™ D Magnetic Particles, 5 x 1 mL
    • Anti-Human CD32 (Fc gamma RII) Blocker, 0.8 mL
  • RoboSep™ Human Myeloid DC Enrichment Kit (Catalog #19061RF)
    • EasySep™ Human Myeloid DC Enrichment Cocktail Component A, 2 x 1 mL
    • EasySep™ Human DC Enrichment Cocktail Component B, 2 x 1 mL
    • EasySep™ D Magnetic Particles, 5 x 1 mL
    • Anti-Human CD32 (Fc gamma RII) Blocker, 0.8 mL
    • RoboSep™ Buffer (Catalog #20104) x 2
    • RoboSep™ Filter Tips (Catalog #20125) x 2
Magnet Compatibility:
• EasySep™ Magnet (Catalog #18000)
• “The Big Easy” EasySep™ Magnet (Catalog #18001)
• 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

Scientific Resources

Educational Materials

(6)

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.

Data and Publications

Data

Start: 1.1% Lin-CD11c+HLA-DR+ of total nucleated cells

Figure 1. Start: 1.1% Lin-CD11c+HLA-DR+ of total nucleated cells

Enriched: 91.6% Lin-CD11c+HLA-DR+ of enriched fraction

Figure 2. Enriched: 91.6% Lin-CD11c+HLA-DR+ of enriched fraction

Starting with 0.6 - 1.8% mDC in fresh peripheral blood nucleated cells, the mDC content of the enriched fraction typically ranges from 79 - 94% purity based on the mDC phenotype of Lineage (CD3, CD14, CD19, CD20, CD34, CD56) negative, HLA-DR positive, and CD11c positive.

Publications

(2)
Blood 2017

NOX5 and p22phox are 2 novel regulators of human monocytic differentiation into dendritic cells.

Marzaioli V et al.

Abstract

Dendritic cells (DCs) are a heterogeneous population of professional antigen-presenting cells and are key cells of the immune system, acquiring different phenotypes in accordance with their localization during the immune response. A subset of inflammatory DCs is derived from circulating monocytes (Mo) and has a key role in inflammation and infection. The pathways controlling Mo-DC differentiation are not fully understood. Our objective was to investigate the possible role of nicotinamide adenine dinucleotide phosphate reduced form oxidases (NOXs) in Mo-DC differentiation. In this study, we revealed that Mo-DC differentiation was inhibited by NOX inhibitors and reactive oxygen species scavengers. We show that the Mo-DC differentiation was dependent on p22phox, and not on gp91phox/NOX2, as shown by the reduced Mo-DC differentiation observed in chronic granulomatous disease patients lacking p22phox. Moreover, we revealed that NOX5 expression was strongly increased during Mo-DC differentiation, but not during Mo-macrophage differentiation. NOX5 was expressed in circulating myeloid DC, and at a lower level in plasmacytoid DC. Interestingly, NOX5 was localized at the outer membrane of the mitochondria and interacted with p22phox in Mo-DC. Selective inhibitors and small interfering RNAs for NOX5 indicated that NOX5 controlled Mo-DC differentiation by regulating the JAK/STAT/MAPK and NFκB pathways. These data demonstrate that the NOX5-p22phox complex drives Mo-DC differentiation, and thus could be critical for immunity and inflammation.
PloS one 2012

Expression profiling of human immune cell subsets identifies miRNA-mRNA regulatory relationships correlated with cell type specific expression.

Allantaz F et al.

Abstract

Blood consists of different cell populations with distinct functions and correspondingly, distinct gene expression profiles. In this study, global miRNA expression profiling was performed across a panel of nine human immune cell subsets (neutrophils, eosinophils, monocytes, B cells, NK cells, CD4 T cells, CD8 T cells, mDCs and pDCs) to identify cell-type specific miRNAs. mRNA expression profiling was performed on the same samples to determine if miRNAs specific to certain cell types down-regulated expression levels of their target genes. Six cell-type specific miRNAs (miR-143; neutrophil specific, miR-125; T cells and neutrophil specific, miR-500; monocyte and pDC specific, miR-150; lymphoid cell specific, miR-652 and miR-223; both myeloid cell specific) were negatively correlated with expression of their predicted target genes. These results were further validated using an independent cohort where similar immune cell subsets were isolated and profiled for both miRNA and mRNA expression. miRNAs which negatively correlated with target gene expression in both cohorts were identified as candidates for miRNA/mRNA regulatory pairs and were used to construct a cell-type specific regulatory network. miRNA/mRNA pairs formed two distinct clusters in the network corresponding to myeloid (nine miRNAs) and lymphoid lineages (two miRNAs). Several myeloid specific miRNAs targeted common genes including ABL2, EIF4A2, EPC1 and INO80D; these common targets were enriched for genes involved in the regulation of gene expression (ptextless9.0E-7). Those miRNA might therefore have significant further effect on gene expression by repressing the expression of genes involved in transcriptional regulation. The miRNA and mRNA expression profiles reported in this study form a comprehensive transcriptome database of various human blood cells and serve as a valuable resource for elucidating the role of miRNA mediated regulation in the establishment of immune cell identity.
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.