(1 reference)-
- 抗原 See all CD25 (IL2RA) products
- CD25 (IL2RA) (Interleukin 2 Receptor, alpha (IL2RA))
- 适用
- 人
- 宿主
- 小鼠
- 克隆类型
- 单克隆
- 标记
- Magnetic Particles
- 应用范围
- Separation (Sep)
- 品牌
- BD IMag™
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- 实验流程
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1. Dilute BD IMag™ Buffer (10X) (Cat. No. 552362) 1:10 with sterile distilled water or prepare 1X BD IMag™ buffer by supplementing Phosphate Buffered Saline with 0.5% BSA, 2 mM EDTA, and 0.09% sodium azide. Store at 4°C.
2. Prepare PBMC from anti-coagulated human blood, preferably by density gradient centrifugation using Ficoll-Paque™. Optional: If Treg cells are desired, enrich the CD4 T lymphocytes by using the BD IMag™ Human CD4 T Lymphocyte Enrichment Set - DM (Cat. no. 557939).
3. Count the cells, wash them with an excess volume of 1X BD IMag™ buffer, and carefully aspirate all the supernatant.
4. Vortex the BD IMag™ Anti-Human CD25 Magnetic Particles - DM thoroughly, and add 50 µl of particles for every 10e7 total cells.
5. MIX THOROUGHLY. Incubate at room temperature for 30 minutes. Avoid nonspecific labeling by working quickly and adhering to recommended incubation times.
6. Bring the BD IMag™-particle labeling volume up to 1-8 x 10e7 cells/ml with 1X BD IMag™ buffer, and immediately place the tube on the BD IMagnet™. Incubate for 8 - 10 minutes.
7. With the tube on the BD IMagnet™, carefully aspirate off the supernatant. This supernatant contains the negative fraction.
8. Remove the tube from the BD IMagnet™, and add 1X BD IMag™ buffer to the same volume as in Step 6. Gently resuspend cells by pipetting up and down, and return the tube to the BD IMagnet™ for another 2 - 4 minutes.
9. With the tube on the BD IMagnet™, carefully aspirate off the supernatant and discard.
10. Repeat Steps 8 and 9.
11. After the final wash step, resuspend the positive fraction in an appropriate buffer or medium, and proceed with desired downstream application(s).
NOTES: Hints for successful cell preparation:
Hints for successful cell preparation:
a) Draw the blood into a tube containing EDTA (for example, BD Vacutainer EDTA tube, Cat. No. 366457 or 367661).
b) Remove the platelet rich plasma by centrifuging once at 220-240 x g.
c) Wash 2-3 times in PBS after the density gradient separation.
d) Remove clumps of cells and/or debris by passing the suspension through a 70-µm nylon cell strainer. - 限制
- 仅限研究用
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- 状态
- Liquid
- 缓冲液
- Aqueous buffered solution containing BSA and ≤0.09 % sodium azide.
- 储存液
- Sodium azide
- 注意事项
- This product contains Sodium azide: a POISONOUS AND HAZARDOUS SUBSTANCE which should be handled by trained staff only.
- 储存条件
- 4 °C
- 储存方法
- Store undiluted at 4°C.
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: "CD4(+) regulatory T cells in autoimmunity and allergy." in: Current opinion in immunology, Vol. 14, Issue 6, pp. 771-8, (2002) (PubMed).
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: "CD4(+) regulatory T cells in autoimmunity and allergy." in: Current opinion in immunology, Vol. 14, Issue 6, pp. 771-8, (2002) (PubMed).
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- 抗原
- CD25 (IL2RA) (Interleukin 2 Receptor, alpha (IL2RA))
- 别名
- CD25 (IL2RA 产品)
- 别名
- CD25 Accessory Reagents, IDDM10 Accessory Reagents, IL2R Accessory Reagents, TCGFR Accessory Reagents, IL2RAC Accessory Reagents, IL-2RA Accessory Reagents, Il2r Accessory Reagents, Ly-43 Accessory Reagents, interleukin 2 receptor subunit alpha Accessory Reagents, interleukin-2 receptor subunit alpha-like Accessory Reagents, interleukin 2 receptor, alpha chain Accessory Reagents, IL2RA Accessory Reagents, Il2ra Accessory Reagents, LOC100222957 Accessory Reagents
- 背景
- BD IMag™ Anti-Human CD25 Magnetic Particles - DM are magnetic nanoparticles that have monoclonal antibody conjugated to their surfaces. These particles are optimized for the positive selection or depletion of CD25-bearing leukocytes using the BD IMagnet™. CD25 is the 55 kDa alpha chain of the IL-2 receptor that is expressed on activated B and T lymphocytes, which may also include regulatory T-cell (Treg cells). For enrichment of Treg cells, depletion of erythrocytes, platelets, monocytes, granulocytes and non-CD4 T lymphocytes is first recommended using the BD IMag™ Human CD4 T Lymphocyte Enrichment Set - DM, followed by the positive selection of the CD25+ population. Peripheral Blood Mononuclear Cells (PBMC) are labeled with BD IMag™ Anti-Human CD25 Magnetic Particles - DM according to the Magnetic Labeling and Separation Protocol. This labeled cell suspension is then placed within the magnetic field of the BD IMagnet™. Labeled cells migrate toward the magnet (positive fraction), leaving the unlabeled cells in suspension so they can be drawn off (negative fraction). The tube is then removed from the magnetic field for the resuspension of the positive fraction. The separation is repeated twice to increase the purity of the positive fraction. The magnetic separation steps are diagrammed in the Separation Flow Chart. After the positive fraction is washed, it can be further evaluated in downstream applications. The small size of the BD IMag™ particles allows the positive fraction to be further evaluated in downstream applications, such as by flow cytometry.
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