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小鼠中性粒细胞分选试剂盒（阴选）是通过阴性分选法从小鼠骨髓或其它组织的单细胞悬液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（非CD11b⁺ Ly-6G⁺ 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，从而达到小鼠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
分选小鼠骨髓、外周血或脾脏中性粒细胞。
| M1522422 | 组件 | 物理外观 | 10T | 50T | 100T | 储存 |
| M1522422A | 生物素标记抗体混合液 | 液体 | 20 μL | 100 μL | 200 μL | 2-8℃. |
| M1522422B | 链霉亲和素磁珠 | 液体 | 200 μL | 1 mL | 1 mL×2 | 2-8℃. |
细胞样品制备
1. 获取细胞与裂解红细胞
获取小鼠骨髓、脾脏或外周血细胞于离心管中，进行红细胞裂解。
注意：红细胞裂解步骤可根据组织样本及所用裂解液的不同调整用量及时间，通常小鼠外周血红细胞裂解需要的时间更长。少量红细胞残留不会影响分选细胞纯度，骨髓细胞不裂解红细胞也可直接进行分选，但分选细胞纯度可能随红细胞占比增加有5%以内小幅度下降。
2. 过滤
裂解完成后，PBS重悬细胞，将细胞悬液用70 μm细胞筛网过滤，500 g，离心5 min。
注意：细胞悬液需要过细胞筛网，以除去组织和细胞团块，否则会影响后续细胞分选纯度。
3. 洗涤细胞
离心完成后，将细胞重悬于分选Buffer，洗涤细胞，500 g离心5 min，弃上清。
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。
4. 调整细胞密度
将细胞重悬于分选buffer中，调整细胞密度为1×10⁸ cells/mL。
分选流程
1. 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1.5 mL离心管底部，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：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。
2. 洗涤细胞1次
孵育完成后，补分选Buffer至1.5 mL，500 g离心5 min。
注意：若分选的细胞数目过多，则使用15 mL离心管，补分选Buffer至5-10 mL后离心。
3. 重悬细胞
离心完成后，弃上清，用100 μL分选Buffer重悬细胞。
注意：如果分选更多细胞，则按比例增加分选buffer用量。
4. 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4°C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。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。
5. 转移与稀释
孵育完成后，转移至流式管中，补分选buffer至2.5 mL，用移液器上下混合充分混匀（避免剧烈振荡或者上下颠倒混匀）。
6. 磁力架分离
将含有细胞的分选流式管置于磁力架上，静置5 min。
7. 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），此细胞悬液中即包含纯化的小鼠中性粒细胞，500 g，离心5 min。离心后弃上清，收集细胞。
8. 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C57BL/6小鼠骨髓细胞中分选中性粒细胞，分选前后的细胞用FITC anti-mouse Ly-6G抗体（克隆号1A8）和PE anti-mouse CD11b抗体（克隆号M1/70）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43.5%和95.1%。
注意事项
磁珠和抗体混合液使用和保存过程中均应避免冷冻。
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。
本产品需与磁性分离器配套使用。
本产品仅供研究使用。
The Mouse Neutrophil Isolation Kit (Negative Isolation) is used to isolate neutrophils from mouse bone marrow or other tissue single-cell suspensions by negative i solation. The principle is to use a cocktail of biotin-labeled monoclonal antibodies against non-target cells (non-CD11b⁺ Ly-6G⁺ cells), followed by removal of these non-target cells using streptavidin-labeled magnetic beads, thereby achieving the isolation of mouse neutrophils. A magnetic separator is required for the isolation process.
Applications
Isolation of neutrophils from mouse bone marrow, peripheral blood, or spleen.
| M1522422 | Component | Appearance | 10T | 50T | 100T | Storage |
| M1522422A | Biotin-Antibody Mix | Liquid | 20 μL | 100 μL | 200 μL | 2-8℃. |
| M1522422B | Streptavidin Magnetic Beads | Liquid | 200 μL | 1 mL | 1 mL×2 | 2-8℃. |
Cell Sample Preparation
Obtain cells and lyse red blood cells
Collect mouse bone marrow, spleen, or peripheral blood cells in a centrifuge tube and perform red blood cell lysis.
Note: The red blood cell lysis step can be adjusted in volume and time depending on the tissue sample and the lysis buffer used. Typically, mouse peripheral blood requires a longer lysis time. A small amount of red blood cell residue will not affect the purity of isolated cells. Bone marrow cells can be processed directly without red blood cell lysis, but the purity of isolated neutrophils may decrease slightly (within 5%) as the proportion of red blood cells increases.
Filter
After lysis, resuspend the cells in PBS, filter the cell suspension through a 70 μm cell strainer, and centrifuge at 500 g for 5 min.
Note: The cell suspension must be passed through a cell strainer to remove tissue and cell clumps; otherwise, subsequent cell isolation purity may be affected.
Wash cells
After centrifugation, resuspend the cells in Isolation Buffer, wash the cells, centrifuge at 500 g for 5 min, and discard the supernatant.
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.
Adjust cell density
Resuspend the cells in Isolation Buffer to a density of 1×10⁸ cells/mL.
Isolation Protocol
Add Biotin-Antibody Mix
Add 100 μL of cell suspension (1×10⁷ cells) to the bottom of a 1.5 mL centrifuge tube, then add 2 μL of Biotin-Antibody Mix. Mix well and incubate at 4°C 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.
Wash cells once
After incubation, add Isolation Buffer to bring the volume to 1.5 mL, and centrifuge at 500 g for 5 min.
Note: If isolating a very large number of cells, use a 15 mL centrifuge tube, add Isolation Buffer to 5-10 mL, then centrifuge.
Resuspend cells
After centrifugation, discard the supernatant and resuspend the cells in 100 μL of Isolation Buffer.
Note: If isolating more cells, increase the volume of Isolation Buffer proportionally.
Add Streptavidin Magnetic Beads
Add 20 μL of washed Streptavidin Magnetic Beads to the centrifuge tube (the beads must be washed before use: vortex 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 and wash the beads once more, 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 4°C 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. If isolating 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.
Transfer and dilute
After incubation, transfer the mixture to a FACS tube, add Isolation Buffer to a total volume of 2.5 mL, and mix thoroughly by pipetting up and down (avoid vigorous shaking or inverting).
Magnetic separation
Place the FACS tube containing the cell suspension on a magnetic separator and let stand for 5 min.
Collect neutrophils
Gently pour the cell suspension into a sterile centrifuge tube (do not remove the FACS tube from the magnetic separator while pouring). This cell suspension contains the purified mouse neutrophils. Centrifuge at 500 g for 5 min, then discard the supernatant and collect the 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.
Isolation Performance
Neutrophils were isolated from C57BL/6 mouse bone marrow cells. Cells before and after isolation were stained with FITC anti-mouse Ly-6G antibody (clone 1A8) and PE anti-mouse CD11b antibody (clone M1/70) and analyzed by flow cytometry. The purity of neutrophils before and after isolation was 43.5% and 95.1%, 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.
| M1522422 | 组件 | 物理外观 | 10T | 50T | 100T | 储存 |
| M1522422A | 生物素标记抗体混合液 | 液体 | 20 μL | 100 μL | 200 μL | 2-8℃. |
| M1522422B | 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 |
|---|---|---|
 ZJ26F0736971 |
分析证书 | M1522422 |
| ZJ26F0636681 |
分析证书 | M1522422 |
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