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小鼠CD4⁺ 细胞分选试剂盒（阳选）适用于从小鼠脾脏细胞或其它组织的单细胞悬液中分选出CD4⁺ 细胞。原理是利用CD4 捕获抗体对CD4⁺ 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小鼠CD4⁺ 细胞。分选得到的CD4⁺ 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
分选小鼠淋巴器官，如脾脏和淋巴结中的CD4⁺ 细胞。
| M1522421 | 组件 | 物理外观 | 10T | 100T | 储存 |
| M1522421A | CD4 捕获抗体 | 液体 | 20 μL | 200 μL | 2-8℃. |
| M1522421B | 可释放磁珠 | 液体 | 200 μL | 1 mL ×2 | 2-8℃. |
| M1522421C | 磁珠释放缓冲液 | 液体 | 4 mL | 40 mL | 2-8℃. |
操作流程（以分选小鼠脾脏CD4⁺细胞为例）
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，500 g，离心5 min。
2. 裂解红细胞
离心结束，弃上清，加入5 mL红细胞裂解液（ACK），室温裂解5 min，再加入20 mL PBS，500 g，离心5 min。
注意：少量红细胞残留不会影响后续分选及细胞纯度。
3. 过滤与计数
离心完成后，弃上清，将脾细胞重悬于PBS，细胞悬液用70 μm细胞筛网过滤后，计数。计数完成后，500 g，离心5 min。
注意：细胞悬液需要用细胞筛网过滤，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，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，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CD4 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 CD4 捕获抗体，混匀后4°C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CD4 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 CD4 捕获抗体。
6. 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。
7. 第一次磁力架分离与清洗
孵育完成后，在流式管中加入分选buffer至2.5 mL，用移液器吹打5次混匀（避免上下颠倒混匀）。将流式管置于磁力架上，静置5 min。
8. 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。
9. 重复清洗
重复步骤8两次（彻底的清洗可以保证后续洗脱高纯度的目的细胞）。
10. 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。
11. 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，补加分选buffer至2.5 mL，吹打混匀，将流式管置于磁力架上，静置5 min。
12. 第二次洗脱
将上清液转移到一个15 mL离心管中备用（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。
13. 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，补加分选buffer至2.5 mL，吹打混匀，将流式管置于磁力架上，静置5 min。
14. 合并细胞并离心
将上清液与第一次洗脱后的细胞上清液混合，500 g，离心5 min，弃上清，即可收集到无磁珠标记的CD4⁺细胞。
15. 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，可用于后续分子生物学或细胞生物学实验。
分选效果
从BALB/c小鼠脾脏细胞中分选CD4⁺细胞，用PE标记的anti-mouse CD4抗体（克隆号RM4-4）染色后进行流式细胞分析，分选前后的CD4⁺细胞纯度分别为13.3%和97.2%。
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 Mouse CD4⁺ Cell Isolation Kit (Positive Isolation) is used to isolate CD4⁺ cells from mouse spleen or other tissue single-cell suspensions by positive i solation. The principle is to label CD4⁺ cells with a CD4 Capture Antibody, then capture the target cells using Releasable Magnetic Beads, and finally dissociate the beads from the cell surface using a Magnetic Beads Release Buffer, thereby obtaining magnetic bead‑free mouse CD4⁺ cells. The isolated CD4⁺ cells can be used in downstream molecular and cell biology experiments.
Applications
Isolation of CD4⁺ cells from mouse lymphoid organs such as spleen and lymph nodes.
| M1522421 | Component | Appearance | 10T | 100T | Storage |
| M1522421A | CD4 Capture Antibody | Liquid | 20 μL | 200 μL | 2-8℃. |
| M1522421B | Releasable Magnetic Beads | Liquid | 200 μL | 1 mL ×2 | 2-8℃. |
| M1522421C | Magnetic Beads Release Buffer | Liquid | 4 mL | 40 mL | 2-8℃. |
Protocol (Using Mouse Spleen CD4⁺ Cell Isolation as an Example)
Prepare single‑cell suspension
Grind the spleen on a 70 μm cell strainer, rinse the strainer with pre‑chilled PBS, collect the cell suspension into a 50 mL centrifuge tube, and 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, lyse at room temperature for 5 min, then add 20 mL PBS and centrifuge at 500 g for 5 min.
Note: A small amount of residual red blood cells will not affect subsequent isolation or cell purity.
Filter and count cells
After centrifugation, discard the supernatant, resuspend the splenocytes in PBS, filter the cell suspension through a 70 μm cell strainer, and count the cells. After counting, centrifuge at 500 g for 5 min.
Note: The cell suspension must be filtered through a cell strainer to remove tissue and cell clumps; otherwise, subsequent cell isolation purity may be affected.
Adjust cell density
After centrifugation, discard the supernatant, 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 CD4 Capture Antibody
Add 500 μL of cell suspension (5×10⁷ cells) to the bottom of a sterile FACS tube, then add 10 μL of CD4 Capture Antibody. Mix well and incubate at 4°C for 15 min.
Note: Add the cell suspension directly to the bottom of the tube, avoiding the tube wall. Depending on the magnetic separator used, a centrifuge tube can also be used for cell isolation. For other cell numbers, adjust the volume of CD4 Capture Antibody proportionally. If isolating fewer than 1×10⁷ cells, adjust the cell suspension volume to 100 μL and add 2 μL of CD4 Capture Antibody.
Add Releasable Magnetic Beads
After incubation, add 100 μL of washed Releasable Magnetic Beads to the FACS tube, mix well, and incubate at 4°C for 15 min (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).
Note: For other cell numbers, adjust the volume of Releasable Magnetic Beads proportionally. If isolating fewer than 1×10⁷ cells, use 20 μL of Releasable Magnetic Beads.
First magnetic separation and wash
After incubation, add Isolation Buffer to the FACS tube to a total volume of 2.5 mL, and mix by pipetting 5 times (avoid inverting the tube). Place the FACS tube on a magnetic separator and let stand for 5 min.
Discard supernatant and resuspend beads
Aspirate and discard the supernatant. Remove the tube from the magnetic separator, quickly add 2 mL of Isolation Buffer, and pipette repeatedly to disperse the beads. Place the tube back on the magnetic separator and let stand for 5 min.
Repeat washing
Repeat step 8 twice (thorough washing ensures high purity of target cells after subsequent release).
10. First elution
After magnetic separation, aspirate and discard the supernatant. Remove the tube from the magnetic separator, quickly add 1 mL of Magnetic Beads Release Buffer to resuspend the beads (avoid letting the beads dry), transfer the bead suspension to a 1.5 mL centrifuge tube, and rotate at room temperature for 10 min.
Note: For other cell numbers, adjust the volume of Magnetic Beads Release Buffer proportionally. If isolating fewer than 1×10⁷ cells, use 200 μL of Magnetic Beads Release Buffer for elution.
Collect first elution cells
After incubation, pipette repeatedly at least 10 times, transfer the bead suspension to a new FACS tube, add Isolation Buffer to 2.5 mL, mix by pipetting, place the tube on a magnetic separator, and let stand for 5 min.
12. Second elution
Transfer the supernatant to a 15 mL centrifuge tube and keep it (the supernatant contains the target cells; do not discard). Quickly resuspend the beads in 1 mL of Magnetic Beads Release Buffer (avoid drying), transfer to a 1.5 mL centrifuge tube, and rotate at room temperature for 10 min.
Collect second elution cells
After incubation, pipette repeatedly at least 10 times, transfer the bead suspension to a new FACS tube, add Isolation Buffer to 2.5 mL, mix by pipetting, place the tube on a magnetic separator, and let stand for 5 min.
Combine cells and centrifuge
Combine the supernatant with the cell supernatant from the first elution, centrifuge at 500 g for 5 min, discard the supernatant, and collect the magnetic bead‑free CD4⁺ 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
CD4⁺ cells were isolated from BALB/c mouse splenocytes. Cells before and after isolation were stained with PE‑labeled anti‑mouse CD4 antibody (clone RM4‑4) and analyzed by flow cytometry. The purity of CD4⁺ cells before and after isolation was 13.3% and 97.2%, respectively.
Precautions
Avoid freezing any component of the kit 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.
| M1522421 | 组件 | 物理外观 | 10T | 100T | 储存 |
| M1522421A | CD4 捕获抗体 | 液体 | 20 μL | 200 μL | 2-8℃. |
| M1522421B | 可释放磁珠 | 液体 | 200 μL | 1 mL ×2 | 2-8℃. |
| M1522421C | 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 |
|---|---|---|
 ZJ26F0737982 |
分析证书 | M1522421 |
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