EasySep™ Release Mouse CD4+CD304+ Regulatory T Cell Isolation Kit
Immunomagnetic positive selection kit using particle release technology
Isolate CD4+CD304+ regulatory T cells (Tregs) from single-cell suspensions of mouse splenocytes or lymph nodes, in less than 45 minutes, using column-free immunomagnetic positive selection. First, CD304+ cells are isolated using EasySep™ Release technology. Then, bound magnetic particles are removed from the EasySep™-isolated CD304+ cells, and unwanted non-Tregs are targeted for depletion. The final isolated fraction contains highly purified CD4+CD304+ cells that express high levels of FOXP3 and CD25 Treg markers and are immediately ready for downstream applications such as flow cytometry, cell culture, or DNA/RNA extraction. An optional protocol allows for the isolation of CD4+CD304- responder T cells in parallel for use in functional studies.
This kit is a first-of-its-kind mouse Treg isolation kit that targets the CD304 surface marker, which, unlike the CD25 Treg marker commonly used for Treg isolation, does not interfere with IL-2 signaling. Following cell isolation with this EasySep™ kit, antibody complexes remain bound to the cell surface and may interact with Brilliant Violet™ antibody conjugates, polyethylene glycol-modified proteins, or other chemically related ligands.
This kit is a first-of-its-kind mouse Treg isolation kit that targets the CD304 surface marker, which, unlike the CD25 Treg marker commonly used for Treg isolation, does not interfere with IL-2 signaling. Following cell isolation with this EasySep™ kit, antibody complexes remain bound to the cell surface and may interact with Brilliant Violet™ antibody conjugates, polyethylene glycol-modified proteins, or other chemically related ligands.
Data Figures
Figure 1. Quick and Easy Protocol to Isolate Particle-Free Mouse CD4+CD304+ Regulatory T Cells and Responder T Cells Using an EasySep™ Magnet
Diagram of the standard protocol to isolate cells in less than 45 minutes using the EasySep™ Magnet (Catalog #18000). For additional information and detailed instructions regarding the EasySep™ regulatory T cell isolation procedure, or the optional mouse CD4+CD304- responder T cell enrichment protocol, please refer to the PIS under the “Protocols and Documentation” section.
Figure 2. EasySep™ Release Mouse CD4+CD304+ Regulatory T Cell Isolation Kit Yields Highly Purified CD4+CD304+ Cells That Express High Levels of FOXP3 and CD25 Treg Markers
(A) Starting with naïve mouse splenocytes, the final isolated fraction is typically 90.4 ± 3.0% for CD4+CD304+ T cells and 71.0 ± 4.6% for CD4+CD304+CD25hiFOXP3+ cells (mean ± SD; using the “Big Easy” EasySep™ Magnet). In the above example, the purities of the start and final isolated fractions are 3.0% and 90.2% for CD4+CD304+ cells; and 1.9% and 73.0% for CD4+CD304+CD25hiFOXP3+ cells, respectively. (B) Starting with naïve mouse lymph nodes, the final isolated fraction is typically 92.1 ± 0.7% for CD4+CD304+ T cells and 78.1 ± 1.4% for CD4+CD304+CD25hiFOXP3+ cells (mean ± SD; using the “Big Easy” EasySep™ Magnet). In the above example, the purities of the start and final isolated fractions are 5.4% and 92.3% for CD4+CD304+ cells; and 4.0% and 77.1% for CD4+CD304+CD25hiFOXP3+ cells, respectively. (C) Starting with naïve mouse splenocytes, the final isolated fraction is typically 94.1 ± 2.9% for CD4+CD304- responder T cells and 90.6 ± 3.0% for CD4+CD304-CD25-FOXP3- cells (mean ± SD; using the “Big Easy” EasySep™ Magnet). In the above example, the purities of the start and final isolated fractions are 11.9% and 95.4% for CD4+CD304- cells; and 11.4% and 94.4% for CD4+CD304-CD25-FOXP3- cells, respectively.
Protocols and Documentation
Find supporting information and directions for use in the Product Information Sheet or explore additional protocols below.
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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.
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Workflow Stages for T Cell Engineering
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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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