The EasySep™ Human CD4+CD127lowCD49d- Regulatory T Cell Enrichment Kit is designed to isolate CD4+CD127lowCD49d- cells from fresh peripheral blood mononuclear cells by negative selection. Unwanted cells are targeted for removal with Tetrameric Antibody Complexes recognizing non-CD4+ T cells, CD127high, CD49d+ cells and dextran-coated magnetic particles. The labeled cells are separated using the EasySep™ magnet without the use of columns. The desired cells are poured off into a new tube.
Cell Type T Cells, T Cells, CD4+, T Cells, Regulatory
Species Human
Sample Source PBMC
Selection Method Negative
Application Cell Isolation
Brand EasySep, RoboSep
Area of Interest Immunology
Data Figures
Figure 1. Typical EasySep™ Human CD4+CD127lowCD49d- Regulatory T Cell Enrichment Profile
Starting with fresh PBMCs, the CD3+CD4+CD127lowCD49d-CD25+FOXP3+ cell content of the enriched fraction typically ranges from 57.4 - 87.4%. Donor to donor variability is unavoidable and may affect the purity of the enriched fraction. In the example above, the purities of the start and enriched fractions are 1.84% and 84.1%, respectively.
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.
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.
A Transendocytosis Model of CTLA-4 Function Predicts Its Suppressive Behavior on Regulatory T Cells Hou TZ et al. The Journal of Immunology 2015
Abstract
Manipulation of the CD28/CTLA-4 pathway is at the heart of a number of immunomodulatory approaches used in both autoimmunity and cancer. Although it is clear that CTLA-4 is a critical regulator of T cell responses, the immunological contexts in which CTLA-4 controls immune responses are not well defined. In this study, we show that whereas CD80/CD86-dependent activation of resting human T cells caused extensive T cell proliferation and robust CTLA-4 expression, in this context CTLA-4 blocking Abs had no impact on the response. In contrast, in settings where CTLA-4(+) cells were present as regulators�
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