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小鼠B细胞分选试剂盒（阴选）是通过阴性分选法从小鼠脾脏、淋巴结或骨髓细胞中分离出B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B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B细胞分选的目的。分选过程需要用到磁力架。
适用范围
分选小鼠脾脏、淋巴结和骨髓中的B细胞。
| M1522410 | 组件 | 物理外观 | 10T | 50T | 100T | 储存 |
| M1522410A | 生物素标记抗体混合液 | 液体 | 20 μL | 100 μL | 200 μL | 2-8℃. |
| M1522410B | 链霉亲和素磁珠 | 液体 | 200 μL | 1 mL | 1 mL×2 | 2-8℃. |
操作流程
1. 制备单细胞悬液
在70 μm细胞筛网上研磨脾脏，以预冷的PBS冲洗细胞筛网，收集细胞悬液于50 mL离心管中。骨髓细胞悬液制备后同样使用70 μm细胞筛网过滤，收取细胞悬液至50 mL离心管中。500 g，离心5 min。
2. 裂解红细胞
离心结束，弃上清，按照每只小鼠加入5 mL红细胞裂解液（ACK），室温裂解5 min，再加入20 mL PBS终止裂解，500 g，离心5 min。
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。少量红细胞残留不会影响分选细胞纯度。
3. 过滤
离心完成后，弃上清，将脾细胞或骨髓细胞重悬于PBS。细胞悬液用70 μm细胞筛网过滤后，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。
4. 调整细胞密度
离心完成后，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，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。
5. 加入生物素标记抗体混合液并洗涤
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℃孵育10 min。孵育完成后，加入1 mL分选buffer，500 g离心5 min。弃上清，向细胞沉淀加入100 μL分选buffer，重悬细胞，转移至无菌流式管。
注意：加入细胞悬液时将细胞加入离心管或流式管底部，避免沿管壁加入。根据所使用磁力架不同也可全程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。
6. 加入 链霉亲和素磁珠
向流式管中加入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：涡旋振荡彻底重悬磁珠，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），混匀后4℃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。
7. 稀释与混匀
孵育完成后，在流式管中加入2.5 mL分选buffer，用移液器上下混合吹打5次混匀（避免剧烈振荡或者上下颠倒混匀）。
8. 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。
9. 收集B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B细胞，500 g，离心5 min。离心后弃上清，收集细胞。
10. 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小鼠骨髓细胞和脾脏细胞中分选B细胞，分选前后的细胞用FITC标记的anti-mouse CD3抗体（克隆号145-2C11）和APC标记的anti-mouse CD19抗体（克隆号6D5）染色后进行流式细胞分析。
骨髓细胞：分选前后B细胞纯度分别为 24.6% 和 95.8%。
脾细胞：分选前后B细胞纯度分别为 62.9% 和 97.8%。
注意事项
磁珠使用和保存过程中应避免冷冻。
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的损耗。
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。
本产品仅供科研使用。
The Mouse B Cell Isolation Kit (Negative Isolation) is used to isolate B cells from mouse spleen, lymph nodes, or bone marrow by negative i solation. The principle is to use a cocktail of biotin-labeled monoclonal antibodies against non-target cells (non-B cells), followed by removal of these non-target cells using streptavidin-labeled magnetic beads, thereby achieving the isolation of mouse B cells. A magnetic separator is required for the isolation process.
Applications
Isolation of B cells from mouse spleen, lymph nodes, and bone marrow.
| M1522410 | Component | Appearance | 10T | 50T | 100T | Storage |
| M1522410A | Biotin-Antibody Mix | Liquid | 20 μL | 100 μL | 200 μL | 2-8℃. |
| M1522410B | Streptavidin Magnetic Beads | Liquid | 200 μL | 1 mL | 1 mL×2 | 2-8℃. |
Protocol
Prepare single-cell suspension
Grind the spleen on a 70 μm cell strainer, rinse the strainer with pre-chilled PBS, and collect the cell suspension into a 50 mL centrifuge tube. For bone marrow, after preparing the cell suspension, filter it through a 70 μm cell strainer and collect into a 50 mL centrifuge tube. Centrifuge at 500 g for 5 min.
Lyse red blood cells
After centrifugation, discard the supernatant. Add 5 mL of ACK red blood cell lysis buffer per mouse, lyse at room temperature for 5 min, then add 20 mL PBS to stop lysis, and centrifuge at 500 g for 5 min.
Note: The red blood cell lysis step can be adjusted in volume and time depending on the tissue sample and the lysis buffer used. A small amount of red blood cell residue will not affect the purity of isolated cells.
Filter
After centrifugation, discard the supernatant and resuspend the splenocytes or bone marrow cells in PBS. Filter the cell suspension through a 70 μm cell strainer, then 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.
Adjust cell density
After centrifugation, 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), 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 and wash
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. After incubation, add 1 mL of Isolation Buffer and centrifuge at 500 g for 5 min. Discard the supernatant, add 100 μL of Isolation Buffer to the cell pellet, resuspend the cells, and transfer to a sterile FACS tube.
Note: When adding the cell suspension, add it to the bottom of the centrifuge tube or FACS tube, avoiding the tube wall. Depending on the magnetic separator used, the entire process can be performed in a centrifuge tube. If isolating more cells, increase the volume of Biotin-Antibody Mix proportionally.
Add Streptavidin Magnetic Beads
Add 20 μL of washed Streptavidin Magnetic Beads to the FACS 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 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 well 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.
Dilute and mix
After incubation, add 2.5 mL of Isolation Buffer to the FACS tube, and mix gently by pipetting up and down 5 times (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 B cells
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 B cells. 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
B cells were isolated from C57BL/6 mouse bone marrow and spleen cells. Cells before and after isolation were stained with FITC anti-mouse CD3 antibody (clone 145-2C11) and APC anti-mouse CD19 antibody (clone 6D5) and analyzed by flow cytometry.
Bone marrow cells: B cell purity before and after isolation was 24.6% and 95.8%, respectively.
Splenocytes: B cell purity before and after isolation was 62.9% and 97.8%, respectively.
Precautions
Avoid freezing the magnetic 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 magnetic 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.
| M1522410 | 组件 | 物理外观 | 10T | 50T | 100T | 储存 |
| M1522410A | 生物素标记抗体混合液 | 液体 | 20 μL | 100 μL | 200 μL | 2-8℃. |
| M1522410B | 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 |
|---|---|---|
 ZJ26F0737981 |
分析证书 | M1522410 |
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