EasySep™ Human CD4+ T Cell Enrichment Kit

Immunomagnetic negative selection kit

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Immunomagnetic negative selection kit
From: 762 USD

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Overview

The EasySep™ Human CD4+ T Cell Enrichment Kit is designed to isolate CD4+ T cells from fresh or previously frozen peripheral mononuclear cells by negative selection. Unwanted cells are targeted for removal with Tetrameric Antibody Complexes recognizing non-CD4+ T cells and dextran-coated magnetic particles. 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 CD4+ T Cell Isolation Kit (17952) which isolates cells in just 8 minutes.
Advantages:
• Fast, easy-to-use and column-free
• Up to 97% purity
• Untouched, viable cells
Components:
  • EasySep™ Human CD4+ T Cell Enrichment Kit (Catalog #19052)
    • EasySep™ Human CD4+ T Cell Enrichment Cocktail, 1 mL
    • EasySep™ D Magnetic Particles, 2 x 1 mL
  • RoboSep™ Human CD4+ T Cell Enrichment Kit with Filter Tips (Catalog #19052RF)
    • EasySep™ Human CD4+ T Cell Enrichment Cocktail, 1 mL
    • EasySep™ D Magnetic Particles, 2 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, CD4+
Species:
Human
Sample Source:
Leukapheresis; PBMC
Selection Method:
Negative
Application:
Cell Isolation
Brand:
EasySep; RoboSep
Area of Interest:
Immunology

Scientific Resources

Educational Materials

(11)

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 CD4+ T Cell Enrichment Kit

Figure 1. FACS Histogram Results Using EasySep™ Human CD4+ T Cell Enrichment Kit

Starting with frozen mononuclear cells, the CD4+ T cell content of the enriched fraction typically ranges from 92% - 97%.

Publications

(36)
The Journal of clinical investigation 2018 JAN

Latent HIV reservoirs exhibit inherent resistance to elimination by CD8+ T cells.

Huang S-H et al.

Abstract

The presence of persistent, latent HIV reservoirs in CD4+ T cells obstructs current efforts to cure infection. The so-called kick-and-kill paradigm proposes to purge these reservoirs by combining latency-reversing agents with immune effectors such as cytotoxic T lymphocytes. Support for this approach is largely based on success in latency models, which do not fully reflect the makeup of latent reservoirs in individuals on long-term antiretroviral therapy (ART). Recent studies have shown that CD8+ T cells have the potential to recognize defective proviruses, which comprise the vast majority of all infected cells, and that the proviral landscape can be shaped over time due to in vivo clonal expansion of infected CD4+ T cells. Here, we have shown that treating CD4+ T cells from ART-treated individuals with combinations of potent latency-reversing agents and autologous CD8+ T cells consistently reduced cell-associated HIV DNA, but failed to deplete replication-competent virus. These CD8+ T cells recognized and potently eliminated CD4+ T cells that were newly infected with autologous reservoir virus, ruling out a role for both immune escape and CD8+ T cell dysfunction. Thus, our results suggest that cells harboring replication-competent HIV possess an inherent resistance to CD8+ T cells that may need to be addressed to cure infection.
Scientific reports 2017 AUG

Combinations of isoform-targeted histone deacetylase inhibitors and bryostatin analogues display remarkable potency to activate latent HIV without global T-cell activation.

Albert BJ et al.

Abstract

Current antiretroviral therapy (ART) for HIV/AIDS slows disease progression by reducing viral loads and increasing CD4 counts. Yet ART is not curative due to the persistence of CD4+ T-cell proviral reservoirs that chronically resupply active virus. Elimination of these reservoirs through the administration of synergistic combinations of latency reversing agents (LRAs), such as histone deacetylase (HDAC) inhibitors and protein kinase C (PKC) modulators, provides a promising strategy to reduce if not eradicate the viral reservoir. Here, we demonstrate that largazole and its analogues are isoform-targeted histone deacetylase inhibitors and potent LRAs. Significantly, these isoform-targeted HDAC inhibitors synergize with PKC modulators, namely bryostatin-1 analogues (bryologs). Implementation of this unprecedented LRA combination induces HIV-1 reactivation to unparalleled levels and avoids global T-cell activation within resting CD4+ T-cells.
Cell reports 2016 OCT

A Cas9 Ribonucleoprotein Platform for Functional Genetic Studies of HIV-Host Interactions in Primary Human T Cells.

Hultquist JF et al.

Abstract

New genetic tools are needed to understand the functional interactions between HIV and human host factors in primary cells. We recently developed a method to edit the genome of primary CD4(+) T cells by electroporation of CRISPR/Cas9 ribonucleoproteins (RNPs). Here, we adapted this methodology to a high-throughput platform for the efficient, arrayed editing of candidate host factors. CXCR4 or CCR5 knockout cells generated with this method are resistant to HIV infection in a tropism-dependent manner, whereas knockout of LEDGF or TNPO3 results in a tropism-independent reduction in infection. CRISPR/Cas9 RNPs can furthermore edit multiple genes simultaneously, enabling studies of interactions among multiple host and viral factors. Finally, in an arrayed screen of 45 genes associated with HIV integrase, we identified several candidate dependency/restriction factors, demonstrating the power of this approach as a discovery platform. This technology should accelerate target validation for pharmaceutical and cell-based therapies to cure HIV infection.
Virology 2016 NOV

Genetic and phenotypic analyses of sequential vpu alleles from HIV-infected IFN-treated patients.

Vanwalscappel B et al.

Abstract

Treatment of HIV-infected patients with IFN-α results in significant, but clinically insufficient, reductions of viremia. IFN induces the expression of several antiviral proteins including BST-2, which inhibits HIV by multiple mechanisms. The viral protein Vpu counteracts different effects of BST-2. We thus asked if Vpu proteins from IFN-treated patients displayed improved anti-BST-2 activities as compared to Vpu from baseline. Deep-sequencing analyses revealed that in five of seven patients treated by IFN-α for a concomitant HCV infection in the absence of antiretroviral drugs, the dominant Vpu sequences differed before and during treatment. In three patients, vpu alleles that emerged during treatment improved virus replication in the presence of IFN-α, and two of them conferred improved virus budding from cells expressing BST-2. Differences were observed for the ability to down-regulate CD4, while all Vpu variants potently down-modulated BST-2 from the cell surface. This report discloses relevant consequences of IFN-treatment on HIV properties.
Proceedings of the National Academy of Sciences of the United States of America 2016 JUL

HIV-1 and HIV-2 exhibit divergent interactions with HLTF and UNG2 DNA repair proteins.

Hrecka K et al.

Abstract

HIV replication in nondividing host cells occurs in the presence of high concentrations of noncanonical dUTP, apolipoprotein B mRNA-editing, enzyme-catalytic, polypeptide-like 3 (APOBEC3) cytidine deaminases, and SAMHD1 (a cell cycle-regulated dNTP triphosphohydrolase) dNTPase, which maintains low concentrations of canonical dNTPs in these cells. These conditions favor the introduction of marks of DNA damage into viral cDNA, and thereby prime it for processing by DNA repair enzymes. Accessory protein Vpr, found in all primate lentiviruses, and its HIV-2/simian immunodeficiency virus (SIV) SIVsm paralogue Vpx, hijack the CRL4(DCAF1) E3 ubiquitin ligase to alleviate some of these conditions, but the extent of their interactions with DNA repair proteins has not been thoroughly characterized. Here, we identify HLTF, a postreplication DNA repair helicase, as a common target of HIV-1/SIVcpz Vpr proteins. We show that HIV-1 Vpr reprograms CRL4(DCAF1) E3 to direct HLTF for proteasome-dependent degradation independent from previously reported Vpr interactions with base excision repair enzyme uracil DNA glycosylase (UNG2) and crossover junction endonuclease MUS81, which Vpr also directs for degradation via CRL4(DCAF1) E3. Thus, separate functions of HIV-1 Vpr usurp CRL4(DCAF1) E3 to remove key enzymes in three DNA repair pathways. In contrast, we find that HIV-2 Vpr is unable to efficiently program HLTF or UNG2 for degradation. Our findings reveal complex interactions between HIV-1 and the DNA repair machinery, suggesting that DNA repair plays important roles in the HIV-1 life cycle. The divergent interactions of HIV-1 and HIV-2 with DNA repair enzymes and SAMHD1 imply that these viruses use different strategies to guard their genomes and facilitate their replication in the host.
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.