RosetteSep™ Human CD8+ T Cell Enrichment Cocktail

Immunodensity negative selection cocktail

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Immunodensity negative selection cocktail
From: 167 USD

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Overview

The RosetteSep™ Human CD8+ T Cell Enrichment Cocktail is designed to isolate CD8+ T cells from whole blood by negative selection. Unwanted cells are targeted for removal with Tetrameric Antibody Complexes recognizing non-CD8+ T cells and glycophorin A on red blood cells (RBCs). When centrifuged over a buoyant density medium such as RosetteSep™ DM-L (Catalog #15705) or Lymphoprep™ (Catalog #07801), the unwanted cells pellet along with the RBCs. The purified CD8+ T cells are present as a highly enriched population at the interface between the plasma and the buoyant density medium.
Advantages:
• Fast and easy-to-use
• Requires no special equipment or training
• Isolated cells are untouched
• Can be combined with SepMate™ for consistent, high-throughput sample processing
Components:
  • RosetteSep™ Human CD8+ T Cell Enrichment Cocktail (Catalog #15023)
    • RosetteSep™ Human CD8+ T Cell Enrichment Cocktail, 2 mL
  • RosetteSep™ Human CD8+ T Cell Enrichment Cocktail (Catalog #15063)
    • RosetteSep™ Human CD8+ T Cell Enrichment Cocktail, 5 x 2 mL
Subtype:
Cell Isolation Kits
Cell Type:
T Cells; T Cells, CD8+
Species:
Human
Sample Source:
Buffy Coat; Whole Blood
Selection Method:
Negative
Application:
Cell Isolation
Brand:
RosetteSep
Area of Interest:
Immunology

Technical Resources

Educational Materials

(7)

Frequently Asked Questions

What is RosetteSep™?

RosetteSep™ is a rapid cell separation procedure for the isolation of purified cells directly from whole blood, without columns or magnets.

How does RosetteSep™ work?

The antibody cocktail crosslinks unwanted cells to red blood cells (RBCs), forming rosettes. The unwanted cells then pellet with the free RBCs when centrifuged over a density centrifugation medium (e.g. Ficoll-Paque™ PLUS, Lymphoprep™).

What factors affect cell recovery?

The temperature of the reagents can affect cell recovery. All reagents should be at room temperature (sample, density centrifugation medium, PBS, centrifuge) before performing the isolations. Layering can also affect recovery so be sure to carefully layer the sample to avoid mixing with the density centrifugation medium as much as possible. Be sure to collect the entire enriched culture without disturbing the RBC pellet. A small amount of density centrifugation medium can be collected without worry.

Which cell samples can RosetteSep™ be used with?

RosetteSep™ can be used with leukapheresis samples, bone marrow or buffy coat, as long as: the concentration of cells does not exceed 5 x 107 per mL (can dilute if necessary); and there are at least 100 RBCs for every nucleated cell (RBCs can be added if necessary).

Can RosetteSep™ be used with previously frozen or cultured cells?

Yes. Cells should be re-suspended at 2 - 5 x 107 cells / mL in PBS + 2% FBS. Fresh whole blood should be added at 250 µL per mL of sample, as a source of red cells.

Can RosetteSep™ be used to enrich progenitors from cord blood?

Yes. Sometimes cord blood contains immature nucleated red cells that have a lower density than mature RBCs. These immature red cells do not pellet over Ficoll™, which can lead to a higher RBC contamination than peripheral blood separations.

Does RosetteSep™ work with mouse cells?

No, but we have developed EasySep™, a magnetic-based cell isolation system which works with mouse and other non-human species.

Which anticoagulant should be used with RosetteSep™?

Peripheral blood should be collected in heparinized Vacutainers. Cord blood should be collected in ACD.

Should the anticoagulant be washed off before using RosetteSep™?

No, the antibody cocktail can be added directly to the sample.
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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 RosetteSep™ Human CD8+ T Cell Enrichment Cocktail

Figure 1. FACS Histogram Results Using RosetteSep™ Human CD8+ T Cell Enrichment Cocktail

Starting with fresh peripheral blood the CD8+ cell content of the enriched fraction typically ranges from 81% - 95%. *Note: Red blood cells were removed by lysis prior to flow cytometry.

Publications

(16)
Cell 2016 SEP

Engineering T Cells with Customized Therapeutic Response Programs Using Synthetic Notch Receptors

Roybal KT et al.

Abstract

Redirecting T cells to attack cancer using engineered chimeric receptors provides powerful new therapeutic capabilities. However, the effectiveness of therapeutic T cells is constrained by the endogenous T cell response: certain facets of natural response programs can be toxic, whereas other responses, such as the ability to overcome tumor immunosuppression, are absent. Thus, the efficacy and safety of therapeutic cells could be improved if we could custom sculpt immune cell responses. Synthetic Notch (synNotch) receptors induce transcriptional activation in response to recognition of user-specified antigens. We show that synNotch receptors can be used to sculpt custom response programs in primary T cells: they can drive a la carte cytokine secretion profiles, biased T cell differentiation, and local delivery of non-native therapeutic payloads, such as antibodies, in response to antigen. SynNotch T cells can thus be used as a general platform to recognize and remodel local microenvironments associated with diverse diseases.
Cell 2016 FEB

Precision Tumor Recognition by T Cells With Combinatorial Antigen-Sensing Circuits.

Roybal KT et al.

Abstract

T cells can be re-directed to kill cancer cells using chimeric antigen receptors (CARs) or T cell receptors (TCRs). This approach, however, is constrained by the rarity of tumor-specific single antigens. Targeting antigens also found on bystander tissues can cause life-threatening adverse effects. A powerful way to enhance ON-target activity of therapeutic T cells is to engineer them to require combinatorial antigens. Here, we engineer a combinatorially activated T cell circuit in which a synthetic Notch receptor for one antigen induces the expression of a CAR for a second antigen. These dual-receptor AND-gate T cells are only armed and activated in the presence of dual antigen tumor cells. These T cells show precise therapeutic discrimination in vivo-sparing single antigen bystander" tumors while efficiently clearing combinatorial antigen "disease" tumors. This type of precision dual-receptor circuit opens the door to immune recognition of a wider range of tumors. VIDEO ABSTRACT."
Cell Reports 2016 APR

Human Cytotoxic T Lymphocytes Form Dysfunctional Immune Synapses with B Cells Characterized by Non-Polarized Lytic Granule Release.

Kabanova A et al.

Abstract

Suppression of the cytotoxic T cell (CTL) immune response has been proposed as one mechanism for immune evasion in cancer. In this study, we have explored the underlying basis for CTL suppression in the context of B cell malignancies. We document that human B cells have an intrinsic ability to resist killing by freshly isolated cytotoxic T cells (CTLs), but are susceptible to lysis by IL-2 activated CTL blasts and CTLs isolated from immunotherapy-treated patients with chronic lymphocytic leukemia (CLL). Impaired killing was associated with the formation of dysfunctional non-lytic immune synapses characterized by the presence of defective linker for activation of T cells (LAT) signaling and non-polarized release of the lytic granules transported by ADP-ribosylation factor-like protein 8 (Arl8). We propose that non-lytic degranulation of CTLs are a key regulatory mechanism of evasion through which B cells may interfere with the formation of functional immune synapses by CTLs.
Human immunology 2011 FEB

Interleukin-21 and cellular activation concurrently induce potent cytotoxic function and promote antiviral activity in human CD8 T cells.

Parmigiani A et al.

Abstract

Infection with human immunodeficiency virus (HIV)-1 induces a progressive deterioration of the immune system that ultimately leads to acquired immune deficiency syndrome (AIDS). Murine models indicate that the common $-chain ($(c))-sharing cytokine interleukin (IL)-21 and its receptor (IL-21R) play a crucial role in maintaining polyfunctional T cell responses during chronic viral infections. Therefore, we analyzed the ability of this cytokine to modulate the properties of human CD8 T cells in comparison with other $(c)-sharing cytokines (IL-2, IL-7, and IL-15). CD8 T cells from healthy volunteers were stimulated in vitro via T cell receptor signals to mimic the heightened status of immune activation of HIV-infected patients. The administration of IL-21 upregulated cytotoxic effector function and the expression of the costimulatory molecule CD28. Notably, this outcome was not accompanied by increased cellular proliferation or activation. Moreover, IL-21 promoted antiviral activity while not inducing HIV-1 replication in vitro. Thus, IL-21 may be a favorable molecule for immunotherapy and a suitable vaccine adjuvant in HIV-infected individuals.
Transplantation 2009 JUL

Regulatory T-cell suppression of CD8+ T-cell-mediated graft-versus-host reaction requires their presence during priming.

Wang X-N et al.

Abstract

BACKGROUND: Despite the promising therapeutic potential of regulatory T cells (Treg) in animal studies of graft-versus-host disease (GVHD), little is known about their effect on human GVHD. Whether Treg are capable of ameliorating GVHD tissue damage has never been demonstrated in humans. It is also unknown whether Treg modulation of GVH histopathologic damage relies on their presence during effector T-cell priming, or whether allogeneic Treg are safe to use clinically. METHODS: To address these questions, we used an in vitro human skin explant GVHD model, which mimics the physiopathology of GVHD. First, donor"-derived CD8 T cells were stimulated with human leukocyte antigen-unmatched "recipient" dendritic cells (priming phase) then primed "donor" CD8 T cells were co-cultured with "recipient" skin to induce GVH tissue damage (effector phase). "Donor"-derived Treg were added at the priming or effector phase of the GVH response. Histopathologic changes in the skin were evaluated using a clinically validated GVHD scoring system. RESULTS: "Donor"-derived Treg significantly reduced the severity of GVH histopathologic damage when present during T-cell priming. In contrast Treg failed to prevent GVH tissue damage when added to the skin co-culture (effector phase) concurrently with primed T cells. Importantly "donor" Treg alone did not induce GVH tissue damage. Delayed Treg addition led to reduced and impaired Treg suppression of CD8 T-cell activation and their cytolytic function. CONCLUSION: "Donor"-derived Treg effectively suppress CD8 T-cell-mediated GVH tissue damage but are critically required during priming of effector T cells. "Donor"-derived Treg seem to be safe and do not induce GVH histopathologic damage."
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.
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