EasySep™ Human CD8 Positive Selection Kit II

Immunomagnetic positive selection kit

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Immunomagnetic positive selection kit
From: 667 USD

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Overview

The EasySep™ Human CD8 Positive Selection Kit II is designed to isolate CD8+ cells from fresh or previously frozen peripheral blood mononuclear cells or washed leukapheresis samples by immunomagnetic positive selection. Desired cells are targeted with antibody complexes recognizing CD8 and magnetic particles. The cocktail also contains an antibody to human Fc receptor to minimize nonspecific binding. Labeled cells are separated using an EasySep™ magnet without the use of columns. Cells of interest remain in the tube while unwanted cells are poured off. The CD8 antigen is expressed on cytotoxic T cells and weakly on a subset of NK cells.

This product replaces the EasySep™ Human CD8 Positive Selection Kit (Catalog #18053) for even faster cell isolations.
Advantages:
• Fast and easy-to-use
• Up to 99% purity
• No columns required
Components:
  • EasySep™ Human CD8 Positive Selection Kit II (Catalog #17853)
    • EasySep™ Human CD8 Positive Selection Cocktail II, 1 mL
    • EasySep™ Dextran RapidSpheres™ 50100, 1 mL
  • RoboSep™ Human CD8 Positive Selection Kit II (Catalog #17853RF)
    • EasySep™ Human CD8 Positive Selection Cocktail II, 1 mL
    • EasySep™ Dextran RapidSpheres™ 50100, 1 mL
    • RoboSep™ Buffer (Catalog #20104)
    • RoboSep™ Filter Tips (Catalog #20125)
Magnet Compatibility:
• EasySep™ Magnet (Catalog #18000)
• “The Big Easy” EasySep™ Magnet (Catalog #18001)
• EasyPlate™ EasySep™ Magnet (Catalog #18102)
• EasyEights™ EasySep™ Magnet (Catalog #18103)
• Easy 50 EasySep™ Magnet (Catalog #18002)
• RoboSep™-S (Catalog #21000)
Subtype:
Cell Isolation Kits
Cell Type:
T Cells; T Cells, CD8+
Species:
Human
Sample Source:
PBMC
Selection Method:
Positive
Application:
Cell Isolation
Brand:
EasySep; RoboSep
Area of Interest:
Immunology

Scientific Resources

Educational Materials

(9)

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

Figure 1. Typical EasySep™ Human CD8 Positive Selection Profile

Starting with a single cell suspension of human PBMCs, the CD8+ cell content of the isolated fraction is typically 96.5 ± 2.4% (mean ± SD using "The Big Easy" EasySep™ Magnet).

Publications

(6)
Science advances 2019 jan

Multiplexed enrichment and genomic profiling of peripheral blood cells reveal subset-specific immune signatures.

M. Reyes et al.

Abstract

Specialized immune cell subsets are involved in autoimmune disease, cancer immunity, and infectious disease through a diverse range of functions mediated by overlapping pathways and signals. However, subset-specific responses may not be detectable in analyses of whole blood samples, and no efficient approach for profiling cell subsets at high throughput from small samples is available. We present a low-input microfluidic system for sorting immune cells into subsets and profiling their gene expression. We validate the system's technical performance against standard subset isolation and library construction protocols and demonstrate the importance of subset-specific profiling through in vitro stimulation experiments. We show the ability of this integrated platform to identify subset-specific disease signatures by profiling four immune cell subsets in blood from patients with systemic lupus erythematosus (SLE) and matched control subjects. The platform has the potential to make multiplexed subset-specific analysis routine in many research laboratories and clinical settings.
Nature communications 2019 feb

PD-1 blockade potentiates HIV latency reversal ex vivo in CD4+ T cells from ART-suppressed individuals.

R. Fromentin et al.

Abstract

HIV persists in latently infected CD4+ T cells during antiretroviral therapy (ART). Immune checkpoint molecules, including PD-1, are preferentially expressed at the surface of persistently infected cells. However, whether PD-1 plays a functional role in HIV latency and reservoir persistence remains unknown. Using CD4+ T cells from HIV-infected individuals, we show that the engagement of PD-1 inhibits viral production at the transcriptional level and abrogates T-cell receptor (TCR)-induced HIV reactivation in latently infected cells. Conversely, PD-1 blockade with the monoclonal antibody pembrolizumab enhances HIV production in combination with the latency reversing agent bryostatin without increasing T cell activation. Our results suggest that the administration of immune checkpoint blockers to HIV-infected individuals on ART may facilitate latency disruption.
Cells 2019 apr

Chronic Hepatitis C Virus Infection Impairs M1 Macrophage Differentiation and Contributes to CD8+ T-Cell Dysfunction.

F. Ahmed et al.

Abstract

Chronic hepatitis C virus (HCV) infection causes generalized CD8+ T cell impairment, not limited to HCV-specific CD8+ T-cells. Liver-infiltrating monocyte-derived macrophages (MDMs) contribute to the local micro-environment and can interact with and influence cells routinely trafficking through the liver, including CD8+ T-cells. MDMs can be polarized into M1 (classically activated) and M2a, M2b, and M2c (alternatively activated) phenotypes that perform pro- and anti-inflammatory functions, respectively. The impact of chronic HCV infection on MDM subset functions is not known. Our results show that M1 cells generated from chronic HCV patients acquire M2 characteristics, such as increased CD86 expression and IL-10 secretion, compared to uninfected controls. In contrast, M2 subsets from HCV-infected individuals acquired M1-like features by secreting more IL-12 and IFN-gamma. The severity of liver disease was also associated with altered macrophage subset differentiation. In co-cultures with autologous CD8+ T-cells from controls, M1 macrophages alone significantly increased CD8+ T cell IFN-gamma expression in a cytokine-independent and cell-contact-dependent manner. However, M1 macrophages from HCV-infected individuals significantly decreased IFN-gamma expression in CD8+ T-cells. Therefore, altered M1 macrophage differentiation in chronic HCV infection may contribute to observed CD8+ T-cell dysfunction. Understanding the immunological perturbations in chronic HCV infection will lead to the identification of therapeutic targets to restore immune function in HCV+ individuals, and aid in the mitigation of associated negative clinical outcomes.
Nature communications 2018 OCT

Human breast tumor-infiltrating CD8+ T cells retain polyfunctionality despite PD-1 expression.

C. A. Egelston et al.

Abstract

Functional CD8+ T cells in human tumors play a clear role in clinical prognosis and response to immunotherapeutic interventions. PD-1 expression in T cells involved in chronic infections and tumors such as melanoma often correlates with a state of T-cell exhaustion. Here we interrogate CD8+ tumor-infiltrating lymphocytes (TILs) from human breast and melanoma tumors to explore their functional state. Despite expression of exhaustion hallmarks, such as PD-1 expression, human breast tumor CD8+ TILs retain robust capacity for production of effector cytokines and degranulation capacity. In contrast, melanoma CD8+ TILs display dramatic reduction of cytokine production and degranulation capacity. We show that CD8+ TILs from human breast tumors can potently kill cancer cells via bi-specific antibodies. Our data demonstrate that CD8+ TILs in human breast tumors retain polyfunctionality, despite PD-1 expression, and suggest that they may be harnessed for effective immunotherapies.
Nature biotechnology 2018 JAN

Multiplexed droplet single-cell RNA-sequencing using natural genetic variation.

Kang HM et al.

Abstract

Droplet single-cell RNA-sequencing (dscRNA-seq) has enabled rapid, massively parallel profiling of transcriptomes. However, assessing differential expression across multiple individuals has been hampered by inefficient sample processing and technical batch effects. Here we describe a computational tool, demuxlet, that harnesses natural genetic variation to determine the sample identity of each droplet containing a single cell (singlet) and detect droplets containing two cells (doublets). These capabilities enable multiplexed dscRNA-seq experiments in which cells from unrelated individuals are pooled and captured at higher throughput than in standard workflows. Using simulated data, we show that 50 single-nucleotide polymorphisms (SNPs) per cell are sufficient to assign 97% of singlets and identify 92% of doublets in pools of up to 64 individuals. Given genotyping data for each of eight pooled samples, demuxlet correctly recovers the sample identity of<99% of singlets and identifies doublets at rates consistent with previous estimates. We apply demuxlet to assess cell-type-specific changes in gene expression in 8 pooled lupus patient samples treated with interferon (IFN)-β and perform eQTL analysis on 23 pooled samples.
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.