EasySep™ Human CD4+ T Cell Enrichment Kit

Immunomagnetic negative selection kit
Catalog #
19052_C
Immunomagnetic negative selection kit
From: 828 USD
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Required Products
  1. EasySep™ Magnet
    EasySep™ Magnet

    Magnet for column-free immunomagnetic separation

  2. EasySep™ Buffer
    EasySep™ Buffer

    Cell separation buffer

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

Product Documentation

Document Type Product Name Catalog # Lot # Language
Document Type
Product Information Sheet
Product Name
EasySep™ Human CD4+ T Cell Enrichment Kit
Catalog #
19052
Lot #
All
Language
English
Document Type
Product Information Sheet
Product Name
RoboSep™ Human CD4+ T Cell Enrichment Kit with Filter Tips
Catalog #
19052RF
Lot #
All
Language
English
Document Type
Safety Data Sheet 1
Product Name
EasySep™ Human CD4+ T Cell Enrichment Kit
Catalog #
19052
Lot #
All
Language
English
Document Type
Safety Data Sheet 2
Product Name
EasySep™ Human CD4+ T Cell Enrichment Kit
Catalog #
19052
Lot #
All
Language
English
Document Type
Safety Data Sheet 1
Product Name
RoboSep™ Human CD4+ T Cell Enrichment Kit with Filter Tips
Catalog #
19052RF
Lot #
All
Language
English
Document Type
Safety Data Sheet 2
Product Name
RoboSep™ Human CD4+ T Cell Enrichment Kit with Filter Tips
Catalog #
19052RF
Lot #
All
Language
English

Educational Materials (10)

Brochure
Tools For Your Immunology Research
Brochure
EasySep™ Cell Separation Technology
Wallchart
Human Immune Cytokines
Wallchart
Frequencies of Cell Types in Human Peripheral Blood
Wallchart
Antigen Processing and Presentation
Wallchart
The Immune Response to HIV Poster
Video
Simultaneous Cell Isolation from Multiple Samples Using the EasyEights™ EasySep™ Magnet
0:57
Simultaneous Cell Isolation from Multiple Samples Using the EasyEights™ EasySep™ Magnet
Video
How EasySep™ Magnetic Cell Separation Technology Works: Fast and Easy Cell Isolation
1:57
How EasySep™ Magnetic Cell Separation Technology Works: Fast and Easy Cell Isolation
Video
How to Isolate PBMCs from Whole Blood Using Density Gradient Centrifugation (Ficoll™ or Lymphoprep™)
1:37
How to Isolate PBMCs from Whole Blood Using Density Gradient Centrifugation (Ficoll™ or Lymphoprep™)
Video
Isolate Cells with a Simple Pour-Off: EasySep™ Cell Separation Technology
1:13
Isolate Cells with a Simple Pour-Off: EasySep™ Cell Separation Technology

Frequently Asked Question

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.
Read More

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 (45)

Nature immunology 2020 nov Functional CRISPR dissection of gene networks controlling human regulatory T cell identity. K. Schumann et al.

Abstract

Human regulatory T (Treg) cells are essential for immune homeostasis. The transcription factor FOXP3 maintains Treg cell identity, yet the complete set of key transcription factors that control Treg cell gene expression remains unknown. Here, we used pooled and arrayed Cas9 ribonucleoprotein screens to identify transcription factors that regulate critical proteins in primary human Treg cells under basal and proinflammatory conditions. We then generated 54,424 single-cell transcriptomes from Treg cells subjected to genetic perturbations and cytokine stimulation, which revealed distinct gene networks individually regulated by FOXP3 and PRDM1, in addition to a network coregulated by FOXO1 and IRF4. We also discovered that HIVEP2, to our knowledge not previously implicated in Treg cell function, coregulates another gene network with SATB1 and is important for Treg cell-mediated immunosuppression. By integrating CRISPR screens and single-cell RNA-sequencing profiling, we have uncovered transcriptional regulators and downstream gene networks in human Treg cells that could be targeted for immunotherapies.
European journal of medicinal chemistry 2020 jan Tuning isoform selectivity and bortezomib sensitivity with a new class of alkenyl indene PDI inhibitor. R. M. Robinson et al.

Abstract

Protein disulfide isomerase (PDI, PDIA1) is an emerging therapeutic target in oncology. PDI inhibitors have demonstrated a unique propensity to selectively induce apoptosis in cancer cells and overcome resistance to existing therapies, although drug candidates have not yet progressed to the stage of clinical development. We recently reported the discovery of lead indene compound E64FC26 as a potent pan-PDI inhibitor that enhances the cytotoxic effects of proteasome inhibitors in panels of Multiple Myeloma (MM) cells and MM mouse models. An extensive medicinal chemistry program has led to the generation of a diverse library of indene-containing molecules with varying degrees of proteasome inhibitor potentiating activity. These compounds were generated by a novel nucleophilic aromatic ring cyclization and dehydration reaction from the precursor ketones. The results provide detailed structure activity relationships (SAR) around this indene pharmacophore and show a high degree of correlation between potency of PDI inhibition and bortezomib (Btz) potentiation in MM cells. Inhibition of PDI leads to ER and oxidative stress characterized by the accumulation of misfolded poly-ubiquitinated proteins and the induction of UPR biomarkers ATF4, CHOP, and Nrf2. This work characterizes the synthesis and SAR of a new chemical class and further validates PDI as a therapeutic target in MM as a single agent and in combination with proteasome inhibitors.
Scientific reports 2019 mar Activation of Peripheral Blood CD4+ T-Cells in IBS is not Associated with Gastrointestinal or Psychological Symptoms. Y. Nasser et al.

Abstract

Immune activation may underlie the pathogenesis of irritable bowel syndrome (IBS), but the evidence is conflicting. We examined whether peripheral CD4+ T-cells from IBS patients demonstrated immune activation and changes in cytokine production. To gain mechanistic insight, we examined whether immune activation correlated with psychological stress and changing symptoms over time. IBS patients (n = 29) and healthy volunteers (HV; n = 29) completed symptom and psychological questionnaires. IBS patients had a significant increase in CD4+ T-cells expressing the gut homing marker integrin beta7 (p = 0.023) and lymphoid marker CD62L (p = 0.026) compared to HV. Furthermore, phytohaemagglutinin stimulated CD4+ T-cells from IBS-D patients demonstrated increased TNFalpha secretion when compared to HV (p = 0.044). Increased psychological scores in IBS did not correlate with TNFalpha production, while stress hormones inhibited cytokine secretion from CD4+ T-cells of HV in vitro. IBS symptoms, but not markers of immune activation, decreased over time. CD4+ T-cells from IBS-D patients exhibit immune activation, but this did not appear to correlate with psychological stress measurements or changing symptoms over time. This could suggest that immune activation is a surrogate of an initial trigger and/or ongoing parallel peripheral mechanisms.
Scientific reports 2019 mar Hexamethylene bisacetamide impairs NK cell-mediated clearance of acute T lymphoblastic leukemia cells and HIV-1-infected T cells that exit viral latency. E. Giuliani et al.

Abstract

The hexamethylene bisacetamide (HMBA) anticancer drug was dismissed due to limited efficacy in leukemic patients but it may re-enter into the clinics in HIV-1 eradication strategies because of its recently disclosed capacity to reactivate latent virus. Here, we investigated the impact of HMBA on the cytotoxicity of natural killer (NK) cells against acute T lymphoblastic leukemia (T-ALL) cells or HIV-1-infected T cells that exit from latency. We show that in T-ALL cells HMBA upmodulated MICB and ULBP2 ligands for the NKG2D activating receptor. In a primary CD4+ T cell-based latency model, HMBA did not reactivate HIV-1, yet enhanced ULBP2 expression on cells harboring virus reactivated by prostratin (PRO). However, HMBA reduced the expression of NKG2D and its DAP10 adaptor in NK cells, hence impairing NKG2D-mediated cytotoxicity and DAP10-dependent response to IL-15 stimulation. Alongside, HMBA dampened killing of T-ALL targets by IL-15-activated NK cells and impaired NK cell-mediated clearance of PRO-reactivated HIV-1+ cells. Overall, our results demonstrate a dominant detrimental effect of HMBA on the NKG2D pathway that crucially controls NK cell-mediated killing of tumors and virus-infected cells, providing one possible explanation for poor clinical outcome in HMBA-treated cancer patients and raising concerns for future therapeutic application of this drug.
Journal of immunology (Baltimore, Md. : 1950) 2019 jun TRAILshort Protects against CD4 T Cell Death during Acute HIV Infection. S. Natesampillai et al.

Abstract

CD4 T cells from HIV-1 infected patients die at excessive rates compared to those from uninfected patients, causing immunodeficiency. We previously identified a dominant negative ligand that antagonizes the TRAIL-dependent pathway of cell death, which we called TRAILshort. Because the TRAIL pathway has been implicated in CD4 T cell death occurring during HIV-1 infection, we used short hairpin RNA knockdown, CRISPR deletion, or Abs specific for TRAILshort to determine the effect of inhibiting TRAILshort on the outcome of experimental acute HIV infection in vitro. Strikingly, all three approaches to TRAILshort deletion/inhibition enhanced HIV-induced death of both infected and uninfected human CD4 T cells. Thus, TRAILshort impacts T cell dynamics during HIV infection, and inhibiting TRAILshort causes more HIV-infected and uninfected bystander cells to die. TRAILshort is, therefore, a host-derived, host-adaptive mechanism to limit the effects of TRAIL-induced cell death. Further studies on the effects of TRAILshort in other disease states are warranted.
Journal of immunology (Baltimore, Md. : 1950) 2019 jul Signaling Cascade through DC-ASGPR Induces Transcriptionally Active CREB for IL-10 Induction and Immune Regulation. C. Gu et al.

Abstract

The types and magnitude of Ag-specific immune responses can be determined by the functional plasticity of dendritic cells (DCs). However, how DCs display functional plasticity and control host immune responses have not been fully understood. In this study, we report that ligation of DC-asialoglycoprotein receptor (DC-ASGPR), a C-type lectin receptor (CLR) expressed on human DCs, resulted in rapid activation of Syk, followed by PLCgamma2 and PKCdelta engagements. However, different from other Syk-coupled CLRs, including Dectin-1, signaling cascade through DC-ASGPR did not trigger NF-kappaB activation. Instead, it selectively activated MAPK ERK1/2 and JNK. Rapid and prolonged phosphorylation of ERK1/2 led to sequential activation of p90RSK and CREB, which consequently bound to IL10 promoter and initiated cytokine expression. In addition, DC-ASGPR ligation activated Akt, which differentially regulated the activities of GSK-3alpha/beta and beta-catenin and further contributed to IL-10 expression. Our observations demonstrate that DC-ASGPR induces IL-10 expression via an intrinsic signaling pathway, which provides a molecular explanation for DC-ASGPR-mediated programing of DCs to control host immune responses.
View All Publications

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