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人CD34⁺ 细胞富集试剂盒（阴选） 是通过阴性分选法从人细胞样品中富集CD34⁺ 细胞。原理是选用不同的生物素（biotin）标记单克隆抗体对非目标细胞（非CD34⁺ 细胞）进行标记，而后通过链霉亲和素（streptavidin）标记的磁珠对非目标细胞进行清除，从而达到富集人CD34⁺ 细胞的目的。分选过程需要用到磁力架。
适用范围
从人脐带血单个核细胞（CBMC）或外周血单个核细胞（PBMC）中富集CD34⁺ 细胞。
| H1522393 | 组件 | 物理外观 | 10T | 50T | 100T | 储存 |
| H1522393A | 生物素标记抗体混合液 | 液体 | 20 μL | 100 μL | 200 μL | 2-8℃. |
| H1522393B | 链霉亲和素磁珠 | 液体 | 200 μL | 1 mL | 1 mL×2 | 2-8℃. |
操作流程
1. 制备人CBMC或PBMC
利用Ficoll密度梯度离心法从人脐带血或外周血中分离单个核细胞，以PBS洗涤细胞，离心后将CBMC或PBMC重悬于分选buffer中，调整细胞密度为1×10⁸ cells/mL。
注意：分选buffer为含有2 mM EDTA和2%胎牛血清（FBS）的PBS，或者含有2 mM EDTA和0.5% BSA的PBS，需预先通过0.22 μm滤膜过滤除菌。
2. 加入生物素标记抗体混合液
将100 μL细胞悬液（1×10⁷个细胞）加入无菌流式管底部，再加入2 μL 生物素标记抗体混合液，混匀后4°C孵育10 min。
注意：将细胞悬液直接加入流式管底部，避免沿流式管管壁加入。根据所使用磁力架不同也可使用离心管进行细胞分选。如果分选更多细胞，则按比例增加生物素标记抗体混合液的用量。
3. 加入链霉亲和素磁珠
孵育完成后，在流式管中加入20 μL 清洗过的链霉亲和素磁珠，混匀后4°C孵育10 min（磁珠使用前需用分选buffer清洗：涡旋振荡彻底重悬磁珠，取实验需要的磁珠至1.5 mL离心管，加入分选buffer至总体积为1 mL，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重悬磁珠，10000 g离心1 min，弃上清。用与起初相同体积的分选buffer重悬磁珠。如取20 μL磁珠进行清洗，则清洗后用20 μL分选buffer重悬）。
注意：如果分选更多细胞，则按比例增加链霉亲和素磁珠用量。例如分选5×10⁷个细胞，在500 μL细胞悬液中加入10 μL 生物素标记抗体混合液和100 μL 链霉亲和素磁珠。如果分选少于1×10⁷个细胞，则将细胞悬液体积补至100 μL，加入2 μL 生物素标记抗体混合液和20 μL 链霉亲和素磁珠。
4. 稀释与混匀
孵育完成后，在流式管中加入2.5 mL分选buffer，用移液器吹打5次混匀。
5. 磁力架分离
将含有细胞的流式管置于磁力架上，静置5 min。
6. 收集CD34⁺细胞
将细胞悬液转移至一个无菌离心管中（流式管不要脱离磁力架），此细胞悬液中即包含富集的人CD34⁺细胞。300 g，离心5 min。弃上清，收集细胞。
注意：可根据实验需要重复步骤2-6一次，进一步提高CD34⁺细胞的富集效果。
7. 重悬备用
根据实验需要洗涤细胞后，将细胞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。
分选效果
从人CBMC中富集CD34⁺细胞，分选前后的细胞用FITC anti-human CD45（克隆号HI30）和PE anti-human CD34抗体（克隆号581）标记后进行流式细胞仪分析。分选前CD34⁺细胞的纯度为 1.2%，第一次分选后CD34⁺细胞的纯度为 13.9%，第二次分选后CD34⁺细胞的纯度为 33.8%
注意事项
1. 磁珠和抗体混合液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和抗体的损耗。
3. 本产品需与磁力架配套使用。
4. 本产品仅供研究使用。
The Human CD34⁺ Cell Enrichment Kit (Negative Selection) is used to enrich CD34⁺ cells from human cell samples by negative selection. The principle is to use a cocktail of biotin-labeled monoclonal antibodies against non-target cells (non-CD34⁺ cells), followed by removal of these non-target cells using streptavidin-labeled magnetic beads, thereby achieving the enrichment of human CD34⁺ cells. A magnetic separator is required for the enrichment process.
Applications
Enrichment of CD34⁺ cells from human cord blood mononuclear cells (CBMCs) or peripheral blood mononuclear cells (PBMCs).
| H1522393 | Component | Appearance | 10T | 50T | 100T | Storage |
| H1522393A | Biotin-Antibody Mix | Liquid | 20 μL | 100 μL | 200 μL | 2-8℃. |
| H1522393B | Streptavidin Magnetic Beads | Liquid | 200 μL | 1 mL | 1 mL×2 | 2-8℃. |
Protocol
Prepare human CBMCs or PBMCs
Isolate mononuclear cells from human cord blood or peripheral blood using Ficoll density gradient centrifugation. Wash the cells with PBS, and after centrifugation, resuspend the CBMCs or PBMCs in Isolation Buffer to a density of 1×10⁸ cells/mL.
Note: Isolation buffer is PBS containing 2 mM EDTA and 2% fetal bovine serum (FBS), or PBS containing 2 mM EDTA and 0.5% BSA. The buffer must be pre-filtered through a 0.22 μm filter.
Add Biotin-Antibody Mix
Add 100 μL of cell suspension (1×10⁷ cells) to the bottom of a sterile FACS tube, then add 2 μL of Biotin-Antibody Mix. Mix well and incubate at 4°C for 10 min.
Note: Add the cell suspension directly to the bottom of the FACS tube, avoiding the tube wall. Depending on the magnetic separator used, a centrifuge tube can also be used for cell isolation. If enriching more cells, increase the volume of Biotin-Antibody Mix proportionally.
Add Streptavidin Magnetic Beads
After incubation, add 20 μL of washed Streptavidin Magnetic Beads to the FACS tube, mix well, and incubate at 4°C for 10 min (the beads must be washed before use: vortex thoroughly to resuspend the beads, transfer the required volume of beads to a 1.5 mL centrifuge tube, add Isolation Buffer to a total volume of 1 mL, 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 to resuspend the beads, centrifuge at 10,000 g for 1 min, and discard the supernatant. Resuspend the beads in the same volume of Isolation Buffer as originally taken. For example, if 20 μL of beads are taken, resuspend them in 20 μL of Isolation Buffer after washing).
Note: If enriching more cells, increase the volume of Streptavidin Magnetic Beads proportionally. For example, to enrich 5×10⁷ cells, add 10 μL of Biotin-Antibody Mix and 100 μL of Streptavidin Magnetic Beads to 500 μL of cell suspension. If enriching fewer than 1×10⁷ cells, adjust the cell suspension volume to 100 μL, then add 2 μL of Biotin-Antibody Mix and 20 μL of Streptavidin Magnetic Beads.
Dilute and mix
After incubation, add 2.5 mL of Isolation Buffer to the FACS tube, and mix by pipetting 5 times.
Magnetic separation
Place the FACS tube containing the cell suspension on a magnetic separator and let stand for 5 min.
Collect CD34⁺ cells
Transfer the cell suspension to a sterile centrifuge tube (do not remove the FACS tube from the magnetic separator). This cell suspension contains the enriched human CD34⁺ cells. Centrifuge at 300 g for 5 min, then discard the supernatant and collect the cells.
Note: If desired, repeat steps 2-6 once to further increase the enrichment of CD34⁺ cells.
Resuspend for further use
Wash the cells as required for your experiment, then resuspend them in the desired buffer or culture medium for subsequent molecular or cell biology experiments.
Enrichment Performance
CD34⁺ cells were enriched from human CBMCs. Cells before and after enrichment were stained with FITC anti-human CD45 (clone HI30) and PE anti-human CD34 antibody (clone 581) and analyzed by flow cytometry.
Purity of CD34⁺ cells before enrichment: 1.2%
Purity after the first enrichment: 13.9%
Purity after the second enrichment: 33.8%
Precautions
Avoid freezing the magnetic beads and antibody mix during use and storage.
Low-retention pipette tips and centrifuge tubes are recommended to avoid loss of beads and antibodies due to adsorption.
This product must be used with a magnetic separator.
For research use only.
| H1522393 | 组件 | 物理外观 | 10T | 50T | 100T | 储存 |
| H1522393A | 生物素标记抗体混合液 | 液体 | 20 μL | 100 μL | 200 μL | 2-8℃. |
| H1522393B | 链霉亲和素磁珠 | 液体 | 200 μL | 1 mL | 1 mL×2 | 2-8℃. |
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| 批号(Lot Number) | 证书类型 | 货号 |
|---|---|---|
 ZJ26F0737980 |
分析证书 | H1522393 |
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