EasySep™ Human CD4+CD127lowCD49d- Regulatory T Cell Enrichment Kit
Immunomagnetic selection of human CD4+CD127lowCD25+ Tregs using particle release technology
Easily isolate highly purified and magnetic particle-free human CD4+CD127lowCD25+ regulatory T cells (Tregs) from fresh or previously frozen human peripheral blood mononuclear cells (PBMCs) or leukapheresis samples by immunomagnetic positive selection, with the EasySep™ Human CD4+CD127lowCD25+ Regulatory T Cell Isolation Kit. Widely used in published research for more than 20 years, EasySep™ combines the specificity of monoclonal antibodies with the simplicity of a column-free magnetic system.
In this EasySep™ positive selection procedure, desired cells are first labeled with antibody complexes recognizing CD25 and magnetic particles called EasySep™ Releasable RapidSpheres™. Unlike traditional magnetic particles, which stay bound to the target cells, these RapidSpheres™ have a releasable feature. After separation using an EasySep™ magnet, bound magnetic particles are removed from the EasySep™-isolated CD25+ cells using a release agent, 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.
For large-scale isolation of CD4+CD127lowCD25+ Tregs, from leukapheresis samples, see the large-format (1x10^10 cells) kit (Catalog #100-1136).
Learn more about how immunomagnetic EasySep™ technology works or how to fully automate immunomagnetic cell isolation with RoboSep™. Alternatively, choose ready-to-use, ethically sourced, primary Human Peripheral Blood CD4+CD25+CD127lowFOXP3+ T cells (Tregs), Frozen isolated with EasySep™ Human CD4+CD127lowCD25+ Regulatory T Cell Isolation Kit. Explore additional products optimized for your workflow, including those for culture media, supplements, antibodies, and more.
In this EasySep™ positive selection procedure, desired cells are first labeled with antibody complexes recognizing CD25 and magnetic particles called EasySep™ Releasable RapidSpheres™. Unlike traditional magnetic particles, which stay bound to the target cells, these RapidSpheres™ have a releasable feature. After separation using an EasySep™ magnet, bound magnetic particles are removed from the EasySep™-isolated CD25+ cells using a release agent, 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.
For large-scale isolation of CD4+CD127lowCD25+ Tregs, from leukapheresis samples, see the large-format (1x10^10 cells) kit (Catalog #100-1136).
Learn more about how immunomagnetic EasySep™ technology works or how to fully automate immunomagnetic cell isolation with RoboSep™. Alternatively, choose ready-to-use, ethically sourced, primary Human Peripheral Blood CD4+CD25+CD127lowFOXP3+ T cells (Tregs), Frozen isolated with EasySep™ Human CD4+CD127lowCD25+ Regulatory T Cell Isolation Kit. Explore additional products optimized for your workflow, including those for culture media, supplements, antibodies, and more.
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Protocols and Documentation
Find supporting information and directions for use in the Product Information Sheet or explore additional protocols below.
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
Educational Materials (2)
Publications (4)
Fc$\gamma$ receptor-mediated cross-linking codefines the immunostimulatory activity of anti-human CD96 antibodies. JCI insight 2022 oct
Abstract
New strategies that augment T cell responses are required to broaden the therapeutic arsenal against cancer. CD96, TIGIT, and CD226 are receptors that bind to a communal ligand, CD155, and transduce either inhibitory or activating signals. The function of TIGIT and CD226 is established, whereas the role of CD96 remains ambiguous. Using a panel of engineered antibodies, we discovered that the T cell stimulatory activity of anti-CD96 antibodies requires antibody cross-linking and is potentiated by Fc$\gamma$ receptors. Thus, soluble Fc silent" anti-CD96 antibodies failed to stimulate human T cells whereas the same antibodies were stimulatory after coating onto plastic surfaces. Remarkably the activity of soluble anti-CD96 antibodies was reinstated by engineering the Fc domain to a human IgG1 isotype and it was dependent on antibody trans-cross-linking by Fc$\gamma$RI. In contrast neither human IgG2 nor variants with increased Fc$\gamma$ receptor IIB binding possessed stimulatory activity. Anti-CD96 antibodies acted directly on T cells and augmented gene expression networks associated with T cell activation leading to proliferation cytokine secretion and resistance to Treg suppression. Furthermore CD96 expression correlated with survival in HPV+ head and neck squamous cell carcinoma and its cross-linking activated tumor-infiltrating T cells thus highlighting the potential of anti-CD96 antibodies in cancer immunotherapy."
Ethanol Intoxication and Burn Injury Increases Intestinal Regulatory T Cell Population and Regulatory T Cell Suppressive Capability. Shock (Augusta, Ga.) 2022 feb
Abstract
Traumatic injuries, such as burn, are often complicated by ethanol intoxication at the time of injury. This leads to a myriad of complications and post-burn pathologies exacerbated by aberrant immune responses. Recent findings suggest that immune cell dysfunction in the gastrointestinal system is particularly important in deleterious outcomes associated with burn injuries. In particular, intoxication at the time of burn injury leads to compromised intestinal T cell responses, which can diminish intestinal immunity and promote bacterial translocation, allowing for increased secondary infections in the injured host and associated sequelae, such as multiple organ failure and sepsis. Regulatory T cells (Treg) have been identified as important mediators of suppressing effector T cell function. Therefore, the goal of this study was to assess the effects of ethanol intoxication and burn injury on Treg populations in small intestinal immune organs. We also evaluated the suppressive capability of Tregs isolated from injured animals. Male C57BL/6 mice were gavaged with 2.9?Šg/kg ethanol before receiving a ˆ¼12.5% total body surface area scald burn. One day after injury, we identified a significant increase in Tregs number in small intestine Peyer's patches (ˆ¼?—1.5) and lamina propria (ˆ¼?—2). Tregs-producing cytokine IL-10 were also increased in both tissues. Finally, Tregs isolated from ethanol and burn-injured mice were able to suppress proliferation of effector T cells to a greater degree than sham vehicle Tregs. This was accompanied by increased levels of IL-10 and decreased levels of pro-proliferative cytokine IL-2 in cultures containing ethanol + burn Tregs compared with sham Tregs. These findings suggest that Treg populations are increased in intestinal tissues 1 day following ethanol intoxication and burn injury. Tregs isolated from ethanol and burn-injured animals also exhibit a greater suppression of effector T cell proliferation, which may contribute to altered T cell responses following injury.
Identification of a CD4+ T cell line with Treg-like activity. Human immunology 2022 apr
Abstract
Regulatory T cells (Tregs) suppress adaptive immunity and inflammation. Although they play a role in suppressing anti-tumor responses, development of therapeutics that target Tregs is limited by their low abundance, heterogeneity, and lack of specific cell surface markers. We isolated human PBMC-derived CD4+ CD25high Foxp3+ Tregs and demonstrate they suppress stimulated CD4+ PBMCs in a cell contact-dependent manner. Because it is not possible to functionally characterize cells after intracellular Foxp3 staining, we identified a human T cell line, MoT, as a model of human Foxp3+ Tregs. Unlike Jurkat T cells, MoT cells share common surface markers consistent with human PBMC-derived Tregs such as: CD4, CD25, GITR, LAG-3, PD-L1, CCR4. PBMC-derived Tregs and MoT cells, but not Jurkat cells, inhibited proliferation of human CD4+PBMCs in a ratio-dependent manner. Transwell membrane separation prevented suppression of stimulated CD4+PBMC proliferation by MoT cells and Tregs, suggesting cell-cell contact is required for suppressive activity. Blocking antibodies against PD-L1, LAG-3, GITR, CCR4, HLA-DR, or CTLA-4 did not reverse the suppressive activity.We show that human PBMC-derived Tregs and MoT cells suppress stimulated CD4+PBMCs in a cell contact-dependent manner, suggesting that a Foxp3+Treg population suppresses immune responses by an uncharacterized cell contact-dependent mechanism.
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