EasySep™ Direct Human CD4+ T Cell Isolation Kit
Immunomagnetic negative selection from whole blood kit
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EasySep™ Direct Human CD4+ T Cell Isolation Kit -
Label for EasySep™ Direct Human CD4+ T Cell Isolation Kit
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Required Products
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Magnet for column-free immunomagnetic separation -
Compatible antibodies for purity assessment of isolated cells
Overview
Isolate CD4+ T cells directly from human whole blood by immunomagnetic negative selection. Desired cells undergo minimal manipulation and are untouched and highly purified.
This product labels RBCs, platelets and unwanted cells with antibody complexes and magnetic particles. The magnetically labeled RBCs, platelets and non-target cells are separated from the untouched CD4+ T cells in an EasySep™ magnet and poured or pipetted into a new tube.
Isolated cells are available for use in downstream uses such as flow cytometry, functional assays or culture.
This product labels RBCs, platelets and unwanted cells with antibody complexes and magnetic particles. The magnetically labeled RBCs, platelets and non-target cells are separated from the untouched CD4+ T cells in an EasySep™ magnet and poured or pipetted into a new tube.
Isolated cells are available for use in downstream uses such as flow cytometry, functional assays or culture.
Advantages:
• > 99.9% RBC depletion without the need for density gradient centrifugation, sedimentation or lysis
• Up to 96% purity of isolated cells
• Fast, easy-to-use and column-free
• Isolated cells are untouched
• Up to 96% purity of isolated cells
• Fast, easy-to-use and column-free
• Isolated cells are untouched
Components:
- EasySep™ Direct Human CD4+ T Cell Isolation Kit (Catalog #19662)
- EasySep™ Direct Human CD4+ T Cell Isolation Cocktail, 2 x 2.5 mL
- EasySep™ Direct RapidSpheres™, 4 x 2.5 mL
- RoboSep™ Human CD4+ T Cell Isolation Kit with Filter Tips (Catalog #19662RF)
- EasySep™ Direct Human CD4+ T Cell Isolation Cocktail, 2 x 2.5 mL
- EasySep™ Direct RapidSpheres™, 4 x 2.5 mL
- RoboSep™ Buffer (Catalog #20104)
- RoboSep™ Filter Tips (Catalog #20125) x 2
Magnet Compatibility:
• EasySep™ Magnet (Catalog #18000)
• “The Big Easy” EasySep™ Magnet (Catalog #18001)
• Easy 50 EasySep™ Magnet (Catalog #18002)
• EasyEights™ EasySep™ Magnet (Catalog #18103)
• RoboSep™-S (Catalog #21000)
Subtype:
Cell Isolation Kits
Cell Type:
T Cells; T Cells, CD4+
Species:
Human
Sample Source:
Whole Blood
Selection Method:
Negative
Application:
Cell Isolation
Brand:
EasySep; RoboSep
Area of Interest:
Immunology
Related Products
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EasySep™ Human CD4+ T Cell Isolation Kit
8-Minute cell isolation kit using immunomagnetic negative selection -
Primary human cells, fresh
Labeling Antibodies
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Anti-Human CD3 Antibody, Clone UCHT1
Mouse monoclonal IgG1 antibody against human, chimpanzee CD3 -
Anti-Human CD4 Antibody, Clone OKT4
Mouse monoclonal IgG2b antibody against human, rhesus, cynomolgus CD4
Scientific Resources
Product Documentation
Document Type
Product Name
Catalog #
Lot #
Language
19662RF
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Educational Materials
(13)
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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.
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™ Direct Human CD4+ T Cell Isolation Profile
Starting with human whole blood from normal healthy donors, the typical CD4+ T cell (CD3+CD4+) content of the non-lysed final isolated fraction is 93.6 ± 2.5% (gated on CD45) or 93.1 ± 2.5% (not gated on CD45). In the example above, the CD4+ T cell (CD3+CD4+) content of the lysed whole blood start sample and non-lysed final isolated fraction is 16.5% and 95.8% (gated on CD45), respectively, or 16.3% and 95.1% (not gated on CD45), respectively. The starting frequency of CD4+ T cells in the non-lysed whole blood start sample is 0.016% (data not shown).
Publications
(2)
Science signaling 2018 MAY
Tuning ITAM multiplicity on T cell receptors can control potency and selectivity to ligand density.
Abstract
Abstract
The T cell antigen receptor (TCR) recognizes peptides from pathogenic proteins bound in the major histocompatibility complex (MHC). To convert this binding event into downstream signaling, the TCR complex contains immunoreceptor tyrosine-based activation motifs (ITAMs) that act as docking sites for the cytoplasmic tyrosine kinase ZAP-70. Unique among antigen receptors, the TCR complex uses 10 ITAMs to transduce peptide-MHC binding to the cell interior. Using synthetic, drug-inducible receptor-ligand pairs, it was found that greater ITAM multiplicity primarily enhanced the efficiency with which ligand binding was converted into an intracellular signal. This manifested as an increase in the fraction of cells that became activated in response to antigen, and a more synchronous initiation of TCR-proximal signaling, rather than direct amplification of the intracellular signals. Exploiting these findings, the potency and selectivity of chimeric antigen receptors targeted against cancer were substantially enhanced by modulating the number of encoded ITAMs.
Nature genetics 2016 DEC
A genome-wide CRISPR screen identifies a restricted set of HIV host dependency factors.
Abstract
Abstract
Host proteins are essential for HIV entry and replication and can be important nonviral therapeutic targets. Large-scale RNA interference (RNAi)-based screens have identified nearly a thousand candidate host factors, but there is little agreement among studies and few factors have been validated. Here we demonstrate that a genome-wide CRISPR-based screen identifies host factors in a physiologically relevant cell system. We identify five factors, including the HIV co-receptors CD4 and CCR5, that are required for HIV infection yet are dispensable for cellular proliferation and viability. Tyrosylprotein sulfotransferase 2 (TPST2) and solute carrier family 35 member B2 (SLC35B2) function in a common pathway to sulfate CCR5 on extracellular tyrosine residues, facilitating CCR5 recognition by the HIV envelope. Activated leukocyte cell adhesion molecule (ALCAM) mediates cell aggregation, which is required for cell-to-cell HIV transmission. We validated these pathways in primary human CD4(+) T cells through Cas9-mediated knockout and antibody blockade. Our findings indicate that HIV infection and replication rely on a limited set of host-dispensable genes and suggest that these pathways can be studied for therapeutic intervention.
PRODUCTS ARE FOR RESEARCH USE ONLY AND NOT INTENDED FOR HUMAN OR ANIMAL DIAGNOSTIC OR THERAPEUTIC USES UNLESS OTHERWISE STATED. FOR ADDITIONAL INFORMATION ON QUALITY AT STEMCELL, REFER TO WWW.STEMCELL.COM/COMPLIANCE.