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.
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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种属
Mouse
Magnet Compatibility
EasySep™ Magnet (Catalog #18000) “The Big Easy” EasySep™ Magnet (Catalog #18001) EasyEights™ EasySep™ Magnet (Catalog #18103) RoboSep™-S (Catalog #21000)
EasySep™小鼠TIL（CD45）正选试剂盒
