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EasySep™ Human CD8+ T Cell Enrichment Kit

Immunomagnetic negative selection kit

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From: 740 USD

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Immunomagnetic negative selection kit
From: 740 USD

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Overview

The EasySep™ Human CD8+ T Cell Enrichment Kit is designed to isolate CD8+ T cells from fresh or previously frozen peripheral blood mononuclear cells or ammonium chloride-lysed leukapheresis sample by negative selection. Unwanted cells are targeted for removal with Tetrameric Antibody Complexes recognizing non-CD8+ T cells and glycophorin A and dextran-coated magnetic particles. The labeled cells are separated using an EasySep™ magnet without the use of columns. Desired cells are poured off into a new tube.

For even faster cell isolations, we recommend the new EasySep™ Human CD8+ T Cell Isolation Kit (17953) which isolates cells in just 8 minutes.
Advantages:
• Fast, easy-to-use and column-free
• Up to 95% purity
• Untouched, viable cells
Components:
  • EasySep™ Human CD8+ T Cell Enrichment Kit (Catalog #19053)
    • EasySep™ Human CD8 T Cell Enrichment Cocktail, 1 mL
    • EasySep™ D Magnetic Particles, 3 x 1 mL
  • RoboSep™ Human CD8+ T Cell Enrichment Kit with Filter Tips (Catalog #19053RF)
    • EasySep™ Human CD8 T Cell Enrichment Cocktail, 1 mL
    • EasySep™ D Magnetic Particles, 3 x 1 mL
    • RoboSep™ Buffer (Catalog #20104)
    • RoboSep™ Filter Tips (Catalog #20125)
Magnet Compatibility:
• EasySep™ Magnet (Catalog #18000)
• “The Big Easy” EasySep™ Magnet (Catalog #18001)
• Easy 50 EasySep™ Magnet (Catalog #18002)
• EasyPlate™ EasySep™ Magnet (Catalog 18102)
• EasyEights™ EasySep™ Magnet (Catalog #18103)
• RoboSep™-S (Catalog #21000)
Subtype:
Cell Isolation Kits
Cell Type:
T Cells; T Cells, CD8+
Species:
Human
Sample Source:
Leukapheresis; PBMC
Selection Method:
Negative
Application:
Cell Isolation
Brand:
EasySep; RoboSep
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

FACS Histogram Results Using EasySep™ Human CD8+ T Cell Enrichment Kit

Figure 1. FACS Histogram Results Using EasySep™ Human CD8+ T Cell Enrichment Kit

Starting with frozen mononuclear cells, the CD8+ cell content of the enriched fraction typically ranges from 84 - 95%

Publications

(10)
Cancer research 2017 NOV

CD155T/TIGIT Signaling Regulates CD8+ T-cell Metabolism and Promotes Tumor Progression in Human Gastric Cancer.

He W et al.

Abstract

The T-cell surface molecule TIGIT is an immune checkpoint molecule that inhibits T-cell responses, but its roles in cancer are little understood. In this study, we evaluated the role TIGIT checkpoint plays in the development and progression of gastric cancer. We show that the percentage of CD8 T cells that are TIGIT+ was increased in gastric cancer patients compared with healthy individuals. These cells showed functional exhaustion with impaired activation, proliferation, cytokine production, and metabolism, all of which were rescued by glucose. In addition, gastric cancer tissue and cell lines expressed CD155, which bound TIGIT receptors and inactivated CD8 T cells. In a T cell-gastric cancer cell coculture system, gastric cancer cells deprived CD8 T cells of glucose and impaired CD8 T-cell effector functions; these effects were neutralized by the additional glucose or by TIGIT blockade. In gastric cancer tumor cells, CD155 silencing increased T-cell metabolism and IFNγ production, whereas CD155 overexpression inhibited T-cell metabolism and IFNγ production; this inhibition was neutralized by TIGIT blockade. Targeting CD155/TIGIT enhanced CD8 T-cell reaction and improved survival in tumor-bearing mice. Combined targeting of TIGIT and PD-1 further enhanced CD8 T-cell activation and improved survival in tumor-bearing mice. Our results suggest that gastric cancer cells inhibit CD8 T-cell metabolism through CD155/TIGIT signaling, which inhibits CD8 T-cell effector functions, resulting in hyporesponsive antitumor immunity. These findings support the candidacy of CD155/TIGIT as a potential therapeutic target in gastric cancer. Cancer Res; 77(22); 6375-88. textcopyright2017 AACR.
Journal of immunology (Baltimore, Md. : 1950) 2016 MAR

Enhanced Cytotoxic CD8 T Cell Priming Using Dendritic Cell-Expressing Human Papillomavirus-16 E6/E7-p16INK4 Fusion Protein with Sequenced Anti-Programmed Death-1.

Garcia-Bates TM et al.

Abstract

The incidence of human papillomavirus (HPV)-related head and neck squamous cell carcinoma has increased in recent decades, though HPV prevention vaccines may reduce this rise in the future. HPV-related cancers express the viral oncoproteins E6 and E7. The latter inactivates the tumor suppressor protein retinoblastoma (Rb), which leads to the overexpression of p16(INK4) protein, providing unique Ags for therapeutic HPV-specific cancer vaccination. We developed potential adenoviral vaccines that express a fusion protein of HPV-16 E6 and E7 (Ad.E6E7) alone or fused with p16 (Ad.E6E7p16) and also encoding an anti-programmed death (PD)-1 Ab. Human monocyte-derived dendritic cells (DC) transduced with Ad.E6E7 or Ad.E6E7p16 with or without Ad.αPD1 were used to activate autologous CD8 CTL in vitro. CTL responses were tested against naturally HPV-infected head and neck squamous cell carcinoma cells using IFN-γ ELISPOT and [(51)Cr]release assay. Surprisingly, stimulation and antitumor activity of CTL were increased after incubation with Ad.E6E7p16-transduced DC (DC.E6E7p16) compared with Ad.E6E7 (DC.E6E7), a result that may be due to an effect of p16 on cyclin-dependent kinase 4 levels and IL-12 secretion by DC. Moreover, the beneficial effect was most prominent when anti-PD-1 was introduced during the second round of stimulation (after initial priming). These data suggest that careful sequencing of Ad.E6E7.p16 with Ad.αPD1 could improve antitumor immunity against HPV-related tumors and that p16 may enhance the immunogenicity of DC, through cyclin-dependent pathways, Th1 cytokine secretion, and by adding a nonviral Ag highly overexpressed in HPV-induced cancers.
PloS one 2015

Application of the pMHC Array to Characterise Tumour Antigen Specific T Cell Populations in Leukaemia Patients at Disease Diagnosis.

Brooks SE et al.

Abstract

Immunotherapy treatments for cancer are becoming increasingly successful, however to further improve our understanding of the T-cell recognition involved in effective responses and to encourage moves towards the development of personalised treatments for leukaemia immunotherapy, precise antigenic targets in individual patients have been identified. Cellular arrays using peptide-MHC (pMHC) tetramers allow the simultaneous detection of different antigen specific T-cell populations naturally circulating in patients and normal donors. We have developed the pMHC array to detect CD8+ T-cell populations in leukaemia patients that recognise epitopes within viral antigens (cytomegalovirus (CMV) and influenza (Flu)) and leukaemia antigens (including Per Arnt Sim domain 1 (PASD1), MelanA, Wilms' Tumour (WT1) and tyrosinase). We show that the pMHC array is at least as sensitive as flow cytometry and has the potential to rapidly identify more than 40 specific T-cell populations in a small sample of T-cells (0.8-1.4 x 10(6)). Fourteen of the twenty-six acute myeloid leukaemia (AML) patients analysed had T cells that recognised tumour antigen epitopes, and eight of these recognised PASD1 epitopes. Other tumour epitopes recognised were MelanA (n = 3), tyrosinase (n = 3) and WT1(126-134) (n = 1). One of the seven acute lymphocytic leukaemia (ALL) patients analysed had T cells that recognised the MUC1(950-958) epitope. In the future the pMHC array may be used provide point of care T-cell analyses, predict patient response to conventional therapy and direct personalised immunotherapy for patients.
Journal of immunology (Baltimore, Md. : 1950) 2010 MAR

Adenosine deaminase modulation of telomerase activity and replicative senescence in human CD8 T lymphocytes.

Parish ST et al.

Abstract

Increased proportions of CD8 T lymphocytes lacking expression of the CD28 costimulatory receptor have been documented during both aging and chronic infection with HIV-1, and their abundance correlates with numerous deleterious clinical outcomes. CD28-negative cells also arise in cell cultures of CD8(+)CD28(+) following multiple rounds of Ag-driven proliferation, reaching the end stage of replicative senescence. The present study investigates the role of a second T cell costimulatory receptor component, adenosine deaminase (ADA), on the process of replicative senescence. We had previously reported that CD28 signaling is required for optimal telomerase upregulation. In this study, we show that the CD8(+)CD28(+) T lymphocytes that are ADA(+) have significantly greater telomerase activity than those that do not express ADA and that ADA is progressively lost as cultures progress to senescence. Because ADA converts adenosine to inosine, cells lacking this enzyme might be subject to prolonged exposure to adenosine, which has immunosuppressive effects. Indeed, we show that chronic exposure of CD8 T lymphocytes to exogenous adenosine accelerates the process of replicative senescence, causing a reduction in overall proliferative potential, reduced telomerase activity, and blunted IL-2 gene transcription. The loss of CD28 expression was accelerated, in part due to adenosine-induced increases in constitutive caspase-3, known to act on the CD28 promoter. These findings provide the first evidence for a role of ADA in modulating the process of replicative senescence and suggest that strategies to enhance this enzyme may lead to novel therapeutic approaches for pathologies associated with increases in senescent CD8 T lymphocytes.
Proceedings of the National Academy of Sciences of the United States of America 2010 FEB

Analysis of CD161 expression on human CD8+ T cells defines a distinct functional subset with tissue-homing properties.

Billerbeck E et al.

Abstract

CD8(+) T lymphocytes play a key role in host defense, in particular against important persistent viruses, although the critical functional properties of such cells in tissue are not fully defined. We have previously observed that CD8(+) T cells specific for tissue-localized viruses such as hepatitis C virus express high levels of the C-type lectin CD161. To explore the significance of this, we examined CD8(+)CD161(+) T cells in healthy donors and those with hepatitis C virus and defined a population of CD8(+) T cells with distinct homing and functional properties. These cells express high levels of CD161 and a pattern of molecules consistent with type 17 differentiation, including cytokines (e.g., IL-17, IL-22), transcription factors (e.g., retinoic acid-related orphan receptor gamma-t, P = 6 x 10(-9); RUNX2, P = 0.004), cytokine receptors (e.g., IL-23R, P = 2 x 10(-7); IL-18 receptor, P = 4 x 10(-6)), and chemokine receptors (e.g., CCR6, P = 3 x 10(-8); CXCR6, P = 3 x 10(-7); CCR2, P = 4 x 10(-7)). CD161(+)CD8(+) T cells were markedly enriched in tissue samples and coexpressed IL-17 with high levels of IFN-gamma and/or IL-22. The levels of polyfunctional cells in tissue was most marked in those with mild disease (P = 0.0006). These data define a T cell lineage that is present already in cord blood and represents as many as one in six circulating CD8(+) T cells in normal humans and a substantial fraction of tissue-infiltrating CD8(+) T cells in chronic inflammation. Such cells play a role in the pathogenesis of chronic hepatitis and arthritis and potentially in other infectious and inflammatory diseases of man.
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