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人中性粒细胞分选试剂盒（阴选）是通过阴性分选法从新鲜外周血中分离出中性粒细胞。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，从而达到人中性粒细胞分选的目的。分选过程需要用到磁力架。
适用范围
分选新鲜人外周血中性粒细胞，分选之前需去除红细胞。
| H1522394 | 组件 | 物理外观 | 10T | 50T | 100T | 储存 |
| H1522394A | 生物素标记抗体混合液 | 液体 | 20 μL | 100 μL | 200 μL | 2-8℃. |
| H1522394B | 链霉亲和素磁珠 | 液体 | 200 μL | 1 mL | 1 mL×2 | 2-8℃. |
样本制备
1. 裂解红细胞
取新鲜抗凝外周血于离心管中，加入红细胞裂解液，进行红细胞裂解。
注意：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。一般建议加入相当于外周血体积3–5倍的红细胞裂解液，在4°C或冰上进行裂解。如一次裂解不充分，可进行第二次裂解。少量红细胞残留通常不会影响后续细胞分选的纯度。
2. 过滤与离心
红细胞裂解完成后，用PBS重悬细胞沉淀，经过细胞筛网过滤后，400 g，离心5 min。
3. 重复洗涤
弃上清，加入PBS重复洗涤一次，以充分去除裂解液及细胞碎片。
4. 调整细胞密度
洗涤完成后，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。
操作流程
1. 加入生物素标记抗体混合液
将100 μL细胞悬液（以1×10⁷个细胞为例）加入1.5 mL离心管底部，随后加入2 μL 生物素标记抗体混合液，充分混匀后室温静置孵育10 min。
注意：加入细胞悬液时将细胞加入离心管底部，避免沿管壁加入。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；如分选少于1×10⁷个细胞，需使用分选Buffer补至100 μL，生物素标记抗体混合液仍使用2 μL。
2. 加入链霉亲和素磁珠
在离心管中加入20 μL 清洗过的链霉亲和素磁珠（磁珠使用前需用分选buffer清洗：涡旋振荡充分重悬磁珠，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），充分混匀后室温静置孵育10 min。
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。
3. 转移与稀释
孵育完成后，将细胞和磁珠混合液转移至流式管中，补分选buffer至2.5 mL，用移液器上下吹打充分混匀。
4. 磁力架分离
将流式管置于磁力架上，静置5 min，使磁珠吸附。
5. 收集中性粒细胞
将细胞悬液轻柔倒入一个无菌离心管中（倾倒过程中流式管不要脱离磁力架，避免吸到磁珠），此细胞悬液中即包含纯化的人中性粒细胞。
6. 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
从人外周血中分选中性粒细胞，分选前后的细胞用AF 647标记的anti-human CD66b抗体（克隆号6/40c）和FITC标记的anti-human CD16抗体（克隆号3G8）标记后进行流式细胞仪分析，分选前后的中性粒细胞纯度分别为58.81%和98.08%。
注意事项
1. 磁珠和抗体混合液使用和保存过程中均应避免冷冻。
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. 本产品需与磁性分离器配套使用。
4. 本产品仅供研究使用。
The Human Neutrophil Isolation Kit (Negative Isolation) is used to isolate neutrophils from fresh peripheral blood by negative i solation. The principle is to use a cocktail of biotin-labeled monoclonal antibodies against non-target cells, followed by removal of these non-target cells using streptavidin-labeled magnetic beads, thereby achieving the isolation of human neutrophils. A magnetic separator is required for the isolation process.
Applications
Isolation of neutrophils from fresh human peripheral blood. Red blood cells must be removed prior to isolation.
| H1522394 | Component | Appearance | 10T | 50T | 100T | Storage |
| H1522394A | Biotin-Antibody Mix | Liquid | 20 μL | 100 μL | 200 μL | 2-8℃. |
| H1522394B | Streptavidin Magnetic Beads | Liquid | 200 μL | 1 mL | 1 mL×2 | 2-8℃. |
Sample Preparation
Lyse red blood cells
Place fresh anticoagulated peripheral blood in a centrifuge tube, add red blood cell lysis buffer, and lyse the red blood cells.
Note: The red blood cell lysis step can be adjusted in volume and time depending on the lysis buffer used. It is generally recommended to add 3–5 times the volume of red blood cell lysis buffer relative to the blood volume and perform lysis at 4°C or on ice. If lysis is incomplete, a second lysis may be performed. A small amount of residual red blood cells usually does not affect the purity of subsequent cell isolation.
Filter and centrifuge
After red blood cell lysis, resuspend the cell pellet in PBS, filter through a cell strainer, and centrifuge at 400 g for 5 min.
Repeat wash
Discard the supernatant, add PBS, and wash once more to thoroughly remove the lysis buffer and cell debris.
Adjust cell density
After washing, discard the supernatant and resuspend the cells 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). The buffer must be pre-filtered through a 0.22 μm filter.
Isolation Protocol
Add Biotin-Antibody Mix
Add 100 μL of cell suspension (using 1×10⁷ cells as an example) to the bottom of a 1.5 mL centrifuge tube, then add 2 μL of Biotin-Antibody Mix. Mix thoroughly and incubate at room temperature for 10 min.
Note: When adding the cell suspension, add it to the bottom of the tube, avoiding the tube wall. If isolating more cells, increase the volume of Biotin-Antibody Mix proportionally. If isolating fewer than 1×10⁷ cells, adjust the volume to 100 μL with Isolation Buffer and still use 2 μL of Biotin-Antibody Mix.
Add Streptavidin Magnetic Beads
Add 20 μL of washed Streptavidin Magnetic Beads to the centrifuge tube (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 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 20 μL of beads are taken, resuspend them in 20 μL of Isolation Buffer after washing). Mix thoroughly and incubate at room temperature for 10 min.
Note: If isolating more cells, increase the volume of Streptavidin Magnetic Beads proportionally. For example, to isolate 5×10⁷ cells, add 10 μL of Biotin-Antibody Mix and 100 μL of Streptavidin Magnetic Beads to 500 μL of cell suspension.
Transfer and dilute
After incubation, transfer the cell and bead mixture to a FACS tube, add Isolation Buffer to a total volume of 2.5 mL, and mix thoroughly by pipetting up and down.
Magnetic separation
Place the FACS tube on a magnetic separator and let stand for 5 min to allow the beads to be captured.
Collect neutrophils
Gently pour the cell suspension into a sterile centrifuge tube (do not remove the FACS tube from the magnetic separator while pouring, and avoid sucking up the beads). This cell suspension contains the purified human neutrophils.
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.
Isolation Performance
Neutrophils were isolated from human peripheral blood. Cells before and after isolation were stained with AF 647-labeled anti-human CD66b antibody (clone 6/40c) and FITC-labeled anti-human CD16 antibody (clone 3G8) and analyzed by flow cytometry. The purity of neutrophils before and after isolation was 58.81% and 98.08%, respectively.
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.
| H1522394 | 组件 组件 | 物理外观 | 10T | 50T | 100T | 储存 |
| H1522394A | 生物素标记抗体混合液 | 液体 | 20 μL | 100 μL | 200 μL | 2-8℃. |
| H1522394B | 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 |
|---|---|---|
 ZJ26F0737122 |
分析证书 | H1522394 |
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