人CD3⁺ 细胞去除磁珠 是通过共价偶联的方式将人CD3抗体与磁珠结合，可以用来从人外周血单个核细胞（PBMC）或经红细胞裂解处理的人外周血白细胞（WBC）样本中快速有效去除CD3⁺ 细胞。
适用范围
去除人PBMC或WBC中的CD3⁺ 细胞。
磁珠用量（悬浮液体积）
| 可分选细胞总量 |
250 μL
| 5 × 10⁸ 个细胞 |
1 mL
| 2 × 10⁹ 个细胞 |
操作流程
当样本为PBMC或WBC时：
1. 制备单细胞悬液
获得人PBMC或人WBC后计数，按照每1×10⁸个细胞数量重悬于450 μL分选Buffer中。
注意：分选Buffer为含有2 mM EDTA和2%胎牛血清（FBS）的PBS（FBS可替换成0.5% BSA或者HSA），需预先通过0.22 μm滤膜过滤除菌。
2. 细胞与磁珠的孵育
将步骤1中制备的450 μL细胞悬液加入1.5 mL离心管底部，再加入清洗好后重悬的磁珠50 μL（人CD3⁺ 细胞去除磁珠使用前需用分选Buffer清洗：取用磁珠时选择合适量程的移液枪，以吹打混匀的方式彻底重悬磁珠，注意吹打时避免产生气泡。吸取实验需要体积的磁珠至1.5 mL离心管，加入1 mL分选Buffer，10000 g离心1 min，或使用磁力架磁吸3 min，弃上清。加入1 mL分选Buffer重复洗涤磁珠1次后，用与取用磁珠时相同体积的分选Buffer重悬磁珠。例如：吸取50 μL磁珠进行清洗，则清洗后用50 μL分选Buffer重悬）。吹打混匀离心管内的液体，室温下使用四维旋转混匀仪混匀孵育30 min。
注意：如果分选其它数量的细胞，可按比例调整细胞体积和人CD3磁珠用量。如分选细胞数量少于5×10⁷个，将细胞体积补至225 μL，加入25 μL人CD3磁珠。
3. 磁分离
孵育完成后，加入分选Buffer将液体体积补至3 mL（可分次将液体从1.5 mL离心管转移至流式管内），用移液器上下混合吹打10次混匀（避免使用剧烈振荡或者上下颠倒混匀的方式）。随后，将流式管置于磁力架上，静置5 min。
4. 目的细胞的富集
磁吸完成后，上清液中包含目的细胞。小心收集上清液，注意不要混入磁珠，500 g，离心5 min。离心后弃上清，收集细胞。
5. 重悬备用
根据实验需要决定是否洗涤细胞，或将细胞离心后重悬于所需缓冲液或培养基中，即可用于后续分子生物学或细胞生物学实验。
去除效果
从人WBC和人PBMC中去除CD3⁺细胞，去除前后的细胞用APC anti-human CD3抗体（克隆号OKT3）和PE anti-human CD4抗体（克隆号RPA-T4）标记后进行流式细胞仪分析。结果如下：
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注意事项
1. 磁珠使用和保存过程中应避免冷冻。
2. 建议选用低吸附移液器吸头和离心管，避免因吸附造成磁珠的损耗。
3. 取用磁珠前选择合适量程的移液枪，以吹打的方式彻底重悬磁珠，注意吹打时避免产生气泡。
4. 本产品仅供科研使用。
Human CD3⁺ Cell Removal Beads are generated by covalently coupling anti‑human CD3 antibody to magnetic beads. They can be used for rapid and efficient removal of CD3⁺ cells from human peripheral blood mononuclear cells (PBMCs) or from red blood cell‑lysed human peripheral blood white blood cells (WBCs).
Applications
Removal of CD3⁺ cells from human PBMCs or WBCs.
| Beads Volume (suspension) | Total Cell Number |
|---|
| 250 μL | 5 × 10⁸ cells | | 1 mL | 2 × 10⁹ cells |
|
Protocol
When the sample is PBMC or WBC:
Prepare single‑cell suspension
Count the obtained human PBMCs or human WBCs. Resuspend 1×10⁸ cells in 450 μL of Isolation Buffer.
Note: Isolation buffer is PBS containing 2 mM EDTA and 2% fetal bovine serum (FBS). FBS may be replaced with 0.5% BSA or HSA. The buffer must be pre‑filtered through a 0.22 μm filter.
Incubate cells with beads
Add the 450 μL cell suspension prepared in step 1 to the bottom of a 1.5 mL centrifuge tube. Then add 50 μL of washed and resuspended Human CD3⁺ Cell Removal Beads (the beads must be washed before use: select a pipette with an appropriate volume range, thoroughly resuspend the beads by pipetting, avoiding bubbles. Transfer the required volume of beads to a 1.5 mL centrifuge tube, add 1 mL of Isolation Buffer, centrifuge at 10,000 g for 1 min or use a magnetic separator for 3 min, then discard the supernatant. Add 1 mL of Isolation Buffer, wash the beads once more, and then resuspend the beads in the same volume of Isolation Buffer as originally taken. For example, if 50 μL of beads are taken, resuspend them in 50 μL of Isolation Buffer after washing). Mix the liquid in the tube by pipetting, and incubate at room temperature for 30 min using a rotator.
Note: For other cell numbers, adjust the cell volume and the amount of Human CD3⁺ Cell Removal Beads proportionally. For fewer than 5×10⁷ cells, adjust the cell suspension volume to 225 μL and add 25 μL of Human CD3⁺ Cell Removal Beads.
Magnetic separation
After incubation, add Isolation Buffer to bring the total volume to 3 mL (the liquid can be transferred from the 1.5 mL centrifuge tube to a FACS tube in several steps). Mix thoroughly by pipetting up and down 10 times (avoid vigorous shaking or inverting). Then place the FACS tube on a magnetic separator and let stand for 5 min.
Collect target cells
After magnetic separation, the target cells are in the supernatant. Carefully collect the supernatant (avoid taking up any beads), centrifuge at 500 g for 5 min, discard the supernatant, and collect the cells.
Resuspend for further use
Wash the cells if required for your experiment, or centrifuge and resuspend them in the desired buffer or culture medium for subsequent molecular or cell biology experiments.
Removal Performance
CD3⁺ cells were removed from human WBCs and human PBMCs. Cells before and after removal were stained with APC anti‑human CD3 antibody (clone OKT3) and PE anti‑human CD4 antibody (clone RPA‑T4) and analyzed by flow cytometry. The results are as follows:
WBCs: CD3⁺ cell purity before and after removal was 16.39% (mean±SD: 19.34%±4.16%) and 0.1% (mean±SD: 0.075%±0.04%), respectively.
PBMCs: CD3⁺ cell purity before and after removal was 53% (mean±SD: 47.51%±9.67%) and 0.28% (mean±SD: 0.42%±0.25%), respectively.
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Precautions
Avoid freezing the beads during use and storage.
Low‑retention pipette tips and centrifuge tubes are recommended to avoid loss of beads due to adsorption.
Before taking the beads, select a pipette with an appropriate volume range, thoroughly resuspend the beads by pipetting, and avoid generating bubbles during pipetting.
For research use only.
ZJ26F0737979
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