# Immunodensity negative selection cocktail

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# Immunodensity negative selection cocktail

From: 216 USD
Catalog #
15023_C
Immunodensity negative selection cocktail

• 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

#### What's Included

• 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
New look, same high quality and support! You may notice that your instrument or reagent packaging looks slightly different from images displayed on the website, or from previous orders. We are updating our look but rest assured, the products themselves and how you should use them have not changed. Learn more

## 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.
Magnet Compatibility

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

## Protocols and Documentation

Find supporting information and directions for use in the Product Information Sheet or explore additional protocols below.

Document Type
Product Name
Catalog #
Lot #
Language
Catalog #
15023, 15063
Lot #
All
Language
English
Document Type
Safety Data Sheet
Catalog #
15023, 15063
Lot #
All
Language
English

## 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.

Research Area
Workflow Stages
Workflow Stages for T Cell Research
Workflow Stages for T Cell Engineering
Workflow Stages for T Cell Therapy Research

Wallchart
Wallchart

# 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.

## Publications (20)

Classification of T-cell activation via autofluorescence lifetime imaging. A. J. Walsh et al. Nature biomedical engineering 2020 jul

#### Abstract

The function of a T cell depends on its subtype and activation state. Here, we show that imaging of the autofluorescence lifetime signals of quiescent and activated T cells can be used to classify the cells. T cells isolated from human peripheral blood and activated in culture using tetrameric antibodies against the surface ligands CD2, CD3 and CD28 showed specific activation-state-dependent patterns of autofluorescence lifetime. Logistic regression models and random forest models classified T cells according to activation state with 97-99{\%} accuracy, and according to activation state (quiescent or activated) and subtype (CD3+CD8+ or CD3+CD4+) with 97{\%} accuracy. Autofluorescence lifetime imaging can be used to non-destructively determine T-cell function.
Translational control of tumor immune escape via the eIF4F-STAT1-PD-L1 axis in melanoma. M. Cerezo et al. Nature medicine 2018 OCT

#### Abstract

Preventing the immune escape of tumor cells by blocking inhibitory checkpoints, such as the interaction between programmed death ligand-1 (PD-L1) and programmed death-1 (PD-1) receptor, is a powerful anticancer approach. However, many patients do not respond to checkpoint blockade. Tumor PD-L1 expression is a potential efficacy biomarker, but the complex mechanisms underlying its regulation are not completely understood. Here, we show that the eukaryotic translation initiation complex, eIF4F, which binds the 5' cap of mRNAs, regulates the surface expression of interferon-$\gamma$-induced PD-L1 on cancer cells by regulating translation of the mRNA encoding the signal transducer and activator of transcription 1 (STAT1) transcription factor. eIF4F complex formation correlates with response to immunotherapy in human melanoma. Pharmacological inhibition of eIF4A, the RNA helicase component of eIF4F, elicits powerful antitumor immune-mediated effects via PD-L1 downregulation. Thus, eIF4A inhibitors, in development as anticancer drugs, may also act as cancer immunotherapies.
Response to Treatment with TNFα Inhibitors in Rheumatoid Arthritis Is Associated with High Levels of GM-CSF and GM-CSF(+) T Lymphocytes. Bystrom J et al. Clinical reviews in allergy & immunology 2017 MAY

#### Abstract

Biologic TNFα inhibitors are a mainstay treatment option for patients with rheumatoid arthritis (RA) refractory to other treatment options. However, many patients either do not respond or relapse after initially responding to these agents. This study was carried out to identify biomarkers that can distinguish responder from non-responder patients before the initiation of treatment. The level of cytokines in plasma and those produced by ex vivo T cells, B cells and monocytes in 97 RA patients treated with biologic TNFα inhibitors was measured before treatment and after 1 and 3 months of treatment by multiplex analyses. The frequency of T cell subsets and intracellular cytokines were determined by flow cytometry. The results reveal that pre-treatment, T cells from patients who went on to respond to treatment with biologic anti-TNFα agents produced significantly more GM-CSF than non-responder patients. Furthermore, immune cells from responder patients produced higher levels of IL-1β, TNFα and IL-6. Cytokine profiling in the blood of patients confirmed the association between high levels of GM-CSF and responsiveness to biologic anti-TNFα agents. Thus, high blood levels of GM-CSF pre-treatment had a positive predictive value of 87.5&percnt; (61.6 to 98.5&percnt; at 95&percnt; CI) in treated RA patients. The study also shows that cells from most anti-TNFα responder patients in the current cohort produced higher levels of GM-CSF and TNFα pre-treatment than non-responder patients. Findings from the current study and our previous observations that non-responsiveness to anti-TNFα is associated with high IL-17 levels suggest that the disease in responder and non-responder RA patients is likely to be driven/sustained by different inflammatory pathways. The use of biomarker signatures of distinct pro-inflammatory pathways could lead to evidence-based prescription of the most appropriate biological therapies for different RA patients.

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