EasySep™ Human CD4+CD127lowCD25+ Regulatory T Cell Isolation Kit

Immunomagnetic cell isolation kit using particle release technology

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EasySep™ Human CD4+CD127lowCD25+ Regulatory T Cell Isolation Kit

Immunomagnetic selection of human CD4+CD127lowCD25+ regulatory T cells using particle release technology

1 x 109 cells
Catalog #18063
1,010 USD

Required Products

Overview

This kit isolates human CD4+CD127lowCD25+ regulatory T cells (Tregs) from fresh peripheral blood mononuclear cells (PBMCs) or leukapheresis samples. First, CD25+ cells are isolated by column-free immunomagnetic positive selection using EasySep™ Releasable RapidSpheres™. Then, bound magnetic particles are removed from the EasySep™-isolated CD25+ cells, and unwanted non-Tregs are targeted for depletion. The final isolated fraction contains highly purified CD4+CD127lowCD25+ cells that express high levels of FOXP3 and are immediately ready for downstream applications. An optional protocol allows for the isolation of CD4+CD25- responder T cells in parallel for use in functional studies. Following cell isolation with this EasySep™ kit, antibody complexes remain bound to the cell surface and may interact with Brilliant Violet™ antibody conjugates, polyethylene glycol-modified proteins, or other chemically related ligands.
Advantages:
• Highly purified human CD4+CD127lowCD25+ Tregs isolated in less than 1 hour
• No-wash removal of EasySep™ Releasable RapidSpheres™
• Optional isolation of CD4+CD25- responder T cells from the same sample
Components:
  • EasySep™ Human CD4+CD127lowCD25+ Regulatory T Cell Isolation Kit (Catalog #18063)
    • EasySep™ Human CD25 Positive Selection Cocktail, 1 mL
    • EasySep™ Human CD127high Depletion Cocktail, 1 mL
    • EasySep™ Human CD4+ T Cell Enrichment Cocktail, 1 mL
    • EasySep™ Releasable RapidSpheres™, 1 mL
    • EasySep™ Dextran RapidSpheres™, 2 x 1 mL
    • EasySep™ Release Buffer, 2 x 1 mL
Magnet Compatibility:
• EasySep™ Magnet (Catalog #18000)
• “The Big Easy” EasySep™ Magnet (Catalog #18001)
• EasyEights™ EasySep™ Magnet (Catalog #18103)
• Easy 50 EasySep™ Magnet (Catalog #18002)
Subtype:
Cell Isolation Kits
Cell Type:
T Cells; T Cells, CD4+; T Cells, Regulatory
Species:
Human
Sample Source:
Leukapheresis; PBMC
Selection Method:
Positive
Application:
Cell Isolation
Brand:
EasySep
Area of Interest:
Immunology

Scientific Resources

Educational Materials

(8)

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

Typical Treg Isolation Using EasySep™ Human CD4+CD127lowCD25+ Regulatory T Cell Isolation Kit
EasySep™ Human CD4+CD127lowCD25+ Regulatory T Cell Isolation Kit Protocol

Starting with fresh or previously frozen PBMCs, the regulatory T cell content (CD4+CD25+FOXP3+) of the isolated fraction is typically 85.0 ± 4.8% (mean ± SD). In the above example, the purities of the start and final isolated fractions are 1.8% and 88.2%, respectively.

EasySep™ Human CD4+CD127lowCD25+ Regulatory T Cell Isolation Kit Protocol

EasySep™ Human CD4+CD127lowCD25+ Regulatory T Cell Isolation Kit Protocol for the Separation of Tregs

Publications

(2)
Journal of immunology (Baltimore, Md. : 1950) 2016 JUL

Netrin-1 Augments Chemokinesis in CD4+ T Cells In Vitro and Elicits a Proinflammatory Response In Vivo.

Boneschansker L et al.

Abstract

Netrin-1 is a neuronal guidance cue that regulates cellular activation, migration, and cytoskeleton rearrangement in multiple cell types. It is a chemotropic protein that is expressed within tissues and elicits both attractive and repulsive migratory responses. Netrin-1 has recently been found to modulate the immune response via the inhibition of neutrophil and macrophage migration. However, the ability of Netrin-1 to interact with lymphocytes and its in-depth effects on leukocyte migration are poorly understood. In this study, we profiled the mRNA and protein expression of known Netrin-1 receptors on human CD4(+) T cells. Neogenin, uncoordinated-5 (UNC5)A, and UNC5B were expressed at low levels in unstimulated cells, but they increased following mitogen-dependent activation. By immunofluorescence, we observed a cytoplasmic staining pattern of neogenin and UNC5A/B that also increased following activation. Using a novel microfluidic assay, we found that Netrin-1 stimulated bidirectional migration and enhanced the size of migratory subpopulations of mitogen-activated CD4(+) T cells, but it had no demonstrable effects on the migration of purified CD4(+)CD25(+)CD127(dim) T regulatory cells. Furthermore, using a short hairpin RNA knockdown approach, we observed that the promigratory effects of Netrin-1 on T effectors is dependent on its interactions with neogenin. In the humanized SCID mouse, local injection of Netrin-1 into skin enhanced inflammation and the number of neogenin-expressing CD3(+) T cell infiltrates. Neogenin was also observed on CD3(+) T cell infiltrates within human cardiac allograft biopsies with evidence of rejection. Collectively, our findings demonstrate that Netrin-1/neogenin interactions augment CD4(+) T cell chemokinesis and promote cellular infiltration in association with acute inflammation in vivo.
PLoS One 2015

The Macrophage Galactose-Type C-Type Lectin (MGL) Modulates Regulatory T Cell Functions

Zizzari IG et al.

Abstract

Regulatory T cells (Tregs) are physiologically designed to prevent autoimmune disease and maintain self-tolerance. In tumour microenvironments, their presence is related to a poor prognosis, and they influence the therapeutic outcome due to their capacity to suppress the immune response by cell-cell contact and to release immunosuppressive cytokines. In this study, we demonstrate that Treg immunosuppressive activity can be modulated by the cross-linking between the CD45RA expressed by Tregs and the C-type lectin MGL. This specific interaction strongly decreases the immunosuppressive activity of Tregs, restoring the proliferative capacity of co-cultured T lymphocytes. This effect can be attributed to changes in CD45RA and TCR signalling through the inhibition of Lck and inactivation of Zap-70, an increase in the Foxp3 methylation status and, ultimately, the reduced production of suppressive cytokines. These results indicate a role of MGL as an immunomodulator within the tumour microenvironment interfering with Treg functions, suggesting its possible use in the design of anticancer vaccines.
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.