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人CD66b⁺ 细胞分选试剂盒（阳选）适用于从人外周血单细胞悬液中分选出CD66b⁺ 细胞。原理是利用CD66b 捕获抗体对CD66b⁺ 细胞进行标记，然后通过可释放磁珠对目标细胞进行捕获，再用磁珠释放缓冲液将磁珠从细胞表面解离，从而得到无磁珠标记的人CD66b⁺ 细胞。CD66b是一种95-100 kDa的糖基磷脂酰肌醇（GPI）连接蛋白，它在中性粒细胞和嗜酸性粒细胞上表达，但在嗜碱性粒细胞和淋巴细胞上不表达。分选得到的CD66b⁺ 细胞可应用于下游的分子生物学和细胞生物学实验。
适用范围
分选人外周血CD66b⁺ 细胞。
| H1522391 | 组件 | 物理外观 | 10T | 100T | 储存 |
| H1522391A | CD66b 捕获抗体 | 液体 | 20 μL | 200 μL | 2-8℃. |
| H1522391B | 可释放磁珠 | 液体 | 200 μL | 1 mL ×2 | 2-8℃. |
| H1522391C | 磁珠释放缓冲液 | 液体 | 4 mL | 40 mL | 2-8℃. |
细胞样品制备流程
1. 裂解红细胞
取新鲜抗凝全血于离心管中，进行红细胞裂解。
注意：推荐使用 EDTA 抗凝全血，红细胞裂解步骤可根据所用裂解液的不同调整用量及时间，若一次裂解不充分，可进行二次裂解。少量红细胞残留不会影响分选细胞纯度。
2. 洗涤细胞
裂解完成后，PBS重悬，洗涤细胞，500 g，离心5 min。
3. 调整细胞密度
离心完成后，弃上清，将细胞重悬于分选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，需预先通过0.22 μm滤膜过滤除菌。
细胞分选流程
1. 加入CD66b 捕获抗体
将500 μL细胞悬液（5×10⁷个细胞）加入无菌流式管底部，再加入10 μL CD66b 捕获抗体，混匀后4°C静置孵育15 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。分选其它数量细胞时可按比例调整CD66b 捕获抗体的用量。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 CD66b 捕获抗体。
2. 加入可释放磁珠
孵育完成后，在流式管中加入100 μL 清洗过的可释放磁珠，混匀后4°C静置孵育15 min（磁珠使用前需用分选buffer清洗：涡旋振荡重悬磁珠，吸取实验需要的磁珠至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次后用与原来相同体积的分选buffer重悬磁珠。如吸取20 μL磁珠进行清洗，则清洗后用20 μL分选buffer重悬）。
注意：分选其它数量细胞时可按比例调整可释放磁珠的用量。如果分选少于1×10⁷个细胞，使用20 μL 可释放磁珠。
3. 第一次磁力架分离
孵育完成后，在流式管中加入分选buffer至2.5 mL，用移液器充分吹打混匀（避免上下颠倒混匀）。将流式管置于磁力架上，静置5 min。
4. 吸弃上清，重悬磁珠
吸出并丢弃上清液。将流式管从磁力架上取下，迅速加入2 mL分选buffer，用移液器反复吹打分散磁珠。将流式管置于磁力架上，静置5 min。
5. 重复清洗
重复步骤4两次（彻底的清洗可以保证后续洗脱高纯度的目的细胞）。
6. 第一次洗脱
磁吸结束后，吸出并丢弃上清液。将流式管从磁力架上取下，迅速加入1 mL 磁珠释放缓冲液充分重悬磁珠，避免磁珠干燥，将磁珠悬液转移至1.5 mL离心管中，室温旋转孵育10 min。
注意：分选其它数量细胞时可按比例调整磁珠释放缓冲液的用量。如果分选少于1×10⁷个细胞，使用200 μL 磁珠释放缓冲液洗脱细胞。
7. 收集第一次洗脱细胞
孵育完成后，用移液器反复吹打至少10次，将磁珠悬液转移至一个新的流式管中，置于磁力架上，静置5 min。
注意：分选较少数量细胞时，可补加分选buffer至1.5 mL后再磁吸。如分选2×10⁷个细胞（400 μL 磁珠释放缓冲液），转移至新流式管后，补加分选buffer至1.5 mL，吹打混匀后再置于磁力架上磁吸。
8. 第二次洗脱
将上清液转移到一个新的流式管中备用（上清中含有目的细胞，不要丢弃）。迅速用1 mL 磁珠释放缓冲液重悬磁珠，避免磁珠干燥，将磁珠悬液转移至1.5 mL离心管中，室温旋转孵育10 min。
9. 收集第二次洗脱细胞
孵育完成后，用移液器反复吹打至少10次，将磁珠悬液转移至一个新的流式管中，置于磁力架上，静置5 min。
10. 去除残留磁珠
将上清液与第一次洗脱后的细胞上清液混合，置于磁力架上，静置5 min，去除残留磁珠。
11. 离心收集细胞
将上清液转移至离心管中，500 g，离心5 min，弃上清，即可收集到无磁珠标记的CD66b⁺细胞。
分选效果
从人外周血细胞中分选CD66b⁺细胞，用AF 647标记的anti-CD66b抗体（克隆号6/40c）染色后进行流式细胞分析，分选前后的CD66b⁺细胞纯度分别为52.62%和99.67%。
注意事项
1. 试剂盒各组分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 CD66b⁺ Cell Isolation Kit (Positive Isolation) is suitable for isolating CD66b⁺ cells from a single-cell suspension of human peripheral blood. The principle is to label CD66b⁺ cells using a CD66b capture antibody, then capture the target cells using releasable magnetic beads. The beads are subsequently dissociated from the cell surface using a Magnetic Beads Release Buffer, yielding human CD66b⁺ cells free of magnetic bead labeling. CD66b is a 95-100 kDa glycosylphosphatidylinositol (GPI)-linked protein that is expressed on neutrophils and eosinophils, but not on basophils or lymphocytes. The isolated CD66b⁺ cells can be used in downstream molecular biology and cell biology experiments.
Scope of Application
Isolation of CD66b⁺ cells from human peripheral blood.
| H1522391 | Component | Appearance | 10T | 100T | Storage |
| H1522391A | CD66b Capture Antibody | Liquid | 20 μL | 200 μL | 2-8℃. |
| H1522391B | Releasable Magnetic Beads | Liquid | 200 μL | 1 mL ×2 | 2-8℃. |
| H1522391C | Magnetic Beads Release Buffer | Liquid | 4 mL | 40 mL | 2-8℃. |
Cell Sample Preparation Procedure
1. Red Blood Cell Lysis
Place fresh anticoagulated whole blood into a centrifuge tube and lyse the red blood cells.
Note: EDTA-anticoagulated whole blood is recommended. Adjust the volume and duration of the red blood cell lysis step according to the lysis buffer used. If lysis is insufficient, repeat the lysis step. The presence of a small number of residual red blood cells will not affect the purity of the isolated cells.
2. Cell Washing
After lysis, resuspend the cells in PBS, wash, and centrifuge at 500 × g for 5 minutes.
3. Adjust Cell Density
After centrifugation, discard the supernatant and resuspend the cells in Isolation Buffer. Adjust the cell density to 1 × 10⁸ cells/mL.
Note: Isolation Buffer is PBS containing 2 mM EDTA and 2% fetal bovine serum (FBS). It must be pre-filtered through a 0.22 μm filter for sterilization.
Cell Isolation Procedure
1. Add CD66b Capture Antibody
Add 500 μL of cell suspension (5 × 10⁷ cells) to the bottom of a sterile flow cytometry tube. Then add 10 μL of CD66b Capture Antibody. Mix well and incubate at 4°C for 15 minutes.
Note: Add the cell suspension directly to the bottom of the flow tube, avoiding adding it along the tube wall. Depending on the magnetic stand used, a centrifuge tube can also be used for cell isolation. For isolating other numbers of cells, scale the volume of CD66b Capture Antibody proportionally. If isolating fewer than 1 × 10⁷ cells, bring the cell suspension volume to 100 μL and add 2 μL of CD66b Capture Antibody.
2. Add Releasable Magnetic Beads
After incubation, add 100 μL of washed Releasable Magnetic Beads to the flow tube. Mix well and incubate at 4°C for 15 minutes (Beads need to be washed before use: vortex to resuspend the beads, transfer the required volume to a 1.5 mL centrifuge tube, add 1 mL of Isolation Buffer, centrifuge at 10,000 × g for 1 minute, or use a magnetic stand for 3 minutes, and discard the supernatant. Add 1 mL of Isolation Buffer and repeat the wash. After the second wash, resuspend the beads in the original volume of Isolation Buffer. For example, if 20 μL of beads are washed, resuspend them in 20 μL of Isolation Buffer).
Note: For isolating other numbers of cells, scale the volume of Releasable Magnetic Beads proportionally. If isolating fewer than 1 × 10⁷ cells, use 20 μL of Releasable Magnetic Beads.
3. First Magnetic Separation
After incubation, add Isolation Buffer to the flow tube to bring the volume to 2.5 mL. Mix thoroughly by pipetting (avoid inverting the tube). Place the flow tube on a magnetic stand and let stand for 5 minutes.
4. Discard Supernatant and Resuspend Beads
Aspirate and discard the supernatant. Remove the flow tube from the magnetic stand, quickly add 2 mL of Isolation Buffer, and pipet repeatedly to disperse the beads. Place the tube back on the magnetic stand and let stand for 5 minutes.
5. Repeat Washing
Repeat Step 4 twice (thorough washing ensures high purity of target cells after subsequent elution).
6. First Elution
After the final magnetic aspiration, aspirate and discard the supernatant. Remove the flow tube from the magnetic stand, quickly add 1 mL of Magnetic Beads Release Buffer, and thoroughly resuspend the beads. Avoid allowing the beads to dry out. Transfer the bead suspension to a 1.5 mL centrifuge tube and incubate with rotation at room temperature for 10 minutes.
Note: For isolating other numbers of cells, scale the volume of Magnetic Beads Release Buffer proportionally. If isolating fewer than 1 × 10⁷ cells, use 200 μL of Magnetic Beads Release Buffer for cell elution.
7. Collect First Eluted Cells
After incubation, pipet repeatedly at least 10 times. Transfer the bead suspension to a new flow tube, place it on a magnetic stand, and let stand for 5 minutes.
Note: When isolating a smaller number of cells, add Isolation Buffer to bring the volume to 1.5 mL before magnetic separation. For example, for 2 × 10⁷ cells (using 400 μL of Magnetic Beads Release Buffer), after transferring to a new flow tube, add Isolation Buffer to 1.5 mL, pipet to mix, then place on the magnetic stand.
8. Second Elution
Transfer the supernatant to a new flow tube and set aside (the supernatant contains the target cells; do not discard). Quickly resuspend the beads in 1 mL of Magnetic Beads Release Buffer, avoiding drying. Transfer the bead suspension to a 1.5 mL centrifuge tube and incubate with rotation at room temperature for 10 minutes.
9. Collect Second Eluted Cells
After incubation, pipet repeatedly at least 10 times. Transfer the bead suspension to a new flow tube, place on a magnetic stand, and let stand for 5 minutes.
10. Remove Residual Beads
Combine the supernatant with the supernatant from the first elution (from Step 7). Place the tube on a magnetic stand and let stand for 5 minutes to remove any residual beads.
11. Collect Cells by Centrifugation
Transfer the supernatant to a centrifuge tube and centrifuge at 500 × g for 5 minutes. Discard the supernatant to collect CD66b⁺ cells free of magnetic bead labeling.
Isolation Performance
CD66b⁺ cells were isolated from human peripheral blood cells. The cells were stained with AF 647-conjugated anti-CD66b antibody (clone 6/40c) and analyzed by flow cytometry. The purity of CD66b⁺ cells before and after isolation was 52.62% and 99.67%, respectively.
Precautions
Avoid freezing all components of the kit during use and storage.
Low-retention pipette tips and centrifuge tubes are recommended to avoid loss of magnetic beads and antibodies due to adsorption.
This product must be used in conjunction with a magnetic stand.
This product is for research use only.
| H1522391 | 组件 | 物理外观 | 10T | 100T | 储存 |
| H1522391A | CD66b 捕获抗体 | 液体 | 20 μL | 200 μL | 2-8℃. |
| H1522391B | 可释放磁珠 | 液体 | 200 μL | 1 mL ×2 | 2-8℃. |
| H1522391C | 磁珠释放缓冲液 | 液体 | 4 mL | 40 mL | 2-8℃. |
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| 批号(Lot Number) | 证书类型 | 货号 |
|---|---|---|
 ZJ26F0737983 |
分析证书 | H1522391 |
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