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Cell Isolation of Spleen Mononuclear Cells
脾脏单核细胞的分离   

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Abstract

This method allows you to isolate different subclass mononuclear cells, like B-cells, T cells, CD4+ and CD8+ T, from mouse spleen. By conjugating cells with specific antibodies and subsequently magnetic beads isolation, using the technique from Miltenyi, this allows a high purity.

Keywords: T cell(T细胞), Purification(净化), Lymphocyte(淋巴细胞), Isolation(隔离)

Materials and Reagents

  1. Antibody
    1. FITC-conjugated anti-CD3 antibody (BD Biosciences, catalog number: 553058 , clone 145-2C11)
    2. PE-conjugated anti-CD19 antibody (BD Biosciences, catalog number: 557399 , clone 1D3)
    3. FITC-conjugated anti-CD4 antibody (BD Biosciences, catalog number: 557667 , clone RM4-5)
    4. PE-conjugated anti-CD8 antibody (BD Biosciences, catalog number: 561095 , clone 53-6.7)

  2. Microbeads
    1. Anti-CD43 microbeads (Miltenyi Biotec, catalog number: 130-049-801 )
    2. Anti-CD90 microbeads (Miltenyi Biotec, catalog number: 130-091-376 )
    3. Anti-CD4 microbeads (Miltenyi Biotec, catalog number: 130-049-201 )
    4. Anti-CD8 microbeads (Miltenyi Biotec, catalog number: 130-091-112 )

  3. Others
    1. 1x phosphate-buffered saline (PBS) (pH 7.2) (Life Technologies, Gibco®, catalog number: 10010-031 )
    2. Bovine serum albumin (BSA) (Life Technologies, InvitrogenTM, catalog number: 15561-020 )
    3. EDTA (Sigma-Aldrich, catalog number: EDS-100G )
    4. Ice-cold separation buffer (see Recipes)

Equipment

  1. Scissors and forceps
  2. MiniMACS separation unit (Miltenyi Biotec, MiniMACS Separator, catalog number: 130-090-312 )
  3. Separation column (Miltenyi Biotec, separation column, Type MS, catalog number: 130-042-201 )
  4. Cell strainer (BD Biosciences, catalog number: 352360 )
  5. Flow cytometer (BD Biosciences, Coulter)
  6. Shaker

Procedure

B-cells, total T cells and/or single positive CD4+ and CD8+ T cells, were purified from spleen cells by magnetic separation with the Mini-MACS system [Miltenyi, 1990] (http://www.miltenyibiotec.com). The scheme illustrates how to manage the procedure.


Figure 1. Schematic view of the experimental strategy using magnetic MACS beads to isolate CD4+ cells. Cells were incubated with CD4-,CD8-,CD90- and CD43-antibody conjugated magnetic beads. The cell suspensions were subjected to column selection and placed in the magnetic separator. Flow-through was discarded. Then the column was washed with separation puffer to increase purity and removed afterwards from the magnetic separator. With a plunger the magnetically labelled cells were flushed out of the column.


  1. Sacrifice mouse and isolate the complete spleen.
  2. Pass spleen through a 100-μm cell strainer to get single cells suspension by crushing with forceps and collecting the cell suspension in 5 ml PBS.
  3. Wash with 1x PBS & centrifuge the cells (100 x g for 5 min), then resuspend spleen cells in 80 μl ice-cold separation buffer per 107 cells. From a normal spleen you will get approximately 8 x 107 cells. The overall operation temperature is room temperature. The buffers should be ice cold.
  4. Add a 20 μl aliquot of antibody-conjugated microbeads per 107 cells incubate for 30 min at 4-8 °C at a shaker. No prewash is needed. The number of cells is depending from the animal.
    The following microbeads were used: anti-CD43 microbeads for negative isolation of resting B cells, anti-CD90 microbeads for positive isolation of total T cells, anti-CD4 and anti-CD8 microbeads for positive isolation of the respective T-cell subset.
  5. Wash the column by putting 5 ml 1x PBS on the top. The liquid passes the column by gravity. Then pipette the labelled cell suspension on top of a separation column, which had been washed three times with separation buffer and placed in the MiniMACS separation unit. Pass the suspension through the column.
  6. In case of negative selection of CD43- B cells the effluent was collected as a B-cell fraction and washed respectively centrifuged three times with 5 ml PBS.
    In case of positive selection of CD90+, CD4+ or CD8+ T-cells the effluent was discarded and the columns were washed twice with 500 μl separation buffer. Subsequently, remove columns from the separator and wash magnetically labelled cells out with 1 ml separation buffer using a plunger.
  7. Wash the respective T-cell fraction three times with medium same as B-cells
  8. Assess the purity of the various cell fractions by Flow cytometry analysis using a Flow cytometer. Stain cells with fluorescein isothiocyanate (FITC)-conjugated anti-CD3 (clone 145-2C11 to detect total T-cells), phycoerythrin (PE)-conjugated anti-CD19 (clone 1D3 to detect B-cells), FITC-conjugated anti-CD4 (clone RM4-5 to detect CD4+ T-cells) and PE-conjugated anti-CD8 (clone 53-6.7 to detect CD8+ T-cells) and analyse for positive cells according to standard procedures.

Recipes

  1. Separation buffer
    1x PBS with 5 mM EDTA and 0.5% BSA

Acknowledgments

The authors thank the German Research Foundation (DFG) and the University Erlangen-Nuremberg for funding. We thank A. von Berg and L. Sologub for excellent technical assistance.

References

  1. Miltenyi, S., Muller, W., Weichel, W. and Radbruch, A. (1990). High gradient magnetic cell separation with MACS. Cytometry 11(2): 231-238.

简介

这种方法允许你从小鼠脾脏中分离不同的亚类单核细胞,如B细胞,T细胞,CD4 +和CD8 + T。 通过使用来自Miltenyi的技术使细胞与特异性抗体缀合并随后磁珠分离,这允许高纯度。

关键字:T细胞, 净化, 淋巴细胞, 隔离

材料和试剂

  1. 抗体
    1. FITC缀合的抗CD3抗体(BD Biosciences,目录号:553058,克隆145-2C11)
    2. PE偶联的抗CD19抗体(BD Biosciences,目录号:557399,克隆1D3)
    3. FITC缀合的抗CD4抗体(BD Biosciences,目录号:557667,克隆RM4-5)
    4. PE缀合的抗CD8抗体(BD Biosciences,目录号:561095,克隆53-6.7)

  2. 微珠
    1. 抗CD43微珠(Miltenyi Biotec,目录号:130-049-801)
    2. 抗CD90微珠(Miltenyi Biotec,目录号:130-091-376)
    3. 抗CD4微珠(Miltenyi Biotec,目录号:130-049-201)
    4. 抗CD8微珠(Miltenyi Biotec,目录号:130-091-112)

  3. 其他
    1. 1×磷酸盐缓冲盐水(PBS)(pH 7.2)(Life Technologies,Gibco ,目录号:10010-031)
    2. 牛血清白蛋白(BSA)(Life Technologies,Invitrogen TM ,目录号:15561-020)
    3. EDTA(Sigma-Aldrich,目录号:EDS-100G)
    4. 冰冷的分离缓冲液(见配方)

设备

  1. 剪刀和镊子
  2. MiniMACS分离单元(Miltenyi Biotec,MiniMACS Separator,目录号:130-090-312)
  3. 分离柱(Miltenyi Biotec,分离柱,类型MS,目录号:130-042-201)
  4. 细胞过滤器(BD Biosciences,目录号:352360)
  5. 流式细胞仪(BD Biosciences,Coulter)
  6. 振动器

程序

通过用Mini-MACS系统进行磁性分离从脾细胞中纯化B细胞,总T细胞和/或单阳性CD4 +和CD8 + T细胞[Miltenyi ,1990]( http://www.miltenyibiotec.com )。该方案说明了如何管理程序。


图1.使用磁性MACS珠分离CD4 +细胞的实验策略的示意图。将细胞与CD4-,CD8-,CD90-和CD43-抗体缀合的磁珠孵育。将细胞悬浮液进行柱选择并置于磁性分离器中。弃去流通液。然后用分离吹扫洗涤柱以提高纯度,然后从磁性分离器中除去。使用柱塞将磁性标记的细胞从柱中冲出。


  1. 牺牲小鼠和隔离完整的脾。
  2. 将脾脏通过100-μm细胞过滤器,通过用镊子压碎并将细胞悬浮液收集在5ml PBS中来获得单细胞悬浮液。
  3. 用1x PBS&离心细胞(100×g,5分钟),然后将脾细胞重悬于80μl冰冷的分离缓冲液/10μL细胞中。从正常脾脏,你会得到约8 x 10 7 细胞。整体工作温度为室温。缓冲液应该是冰冷的
  4. 加入20μl等分的抗体结合的微珠/10 10个细胞在4-8℃下在振荡器中温育30分钟。不需要预洗。细胞的数量取决于动物。
    使用以下微珠:用于阴性分离静止B细胞的抗CD43微珠,用于总T细胞的阳性分离的抗CD90微珠,抗CD4和抗CD8微珠,用于相应T细胞亚群的阳性分离。
  5. 通过放置5毫升1 x PBS在顶部洗涤柱。液体通过重力流过柱。然后吸取标记的细胞悬浮液在分离柱顶部,其已用分离缓冲液洗涤三次并置于MiniMACS分离单元中。将悬浮液通过色谱柱。
  6. 在CD43-B细胞的阴性选择的情况下,收集流出物作为B细胞级分,并分别用5ml PBS离心洗涤三次。
    在CD90 +,CD4 +或CD8 + T细胞的阳性选择的情况下,弃去流出物并用500μl分离缓冲液洗涤柱两次。随后,从分离器中取出柱,并使用柱塞用1ml分离缓冲液洗涤磁性标记的细胞。
  7. 用与B细胞相同的培养基洗涤各自的T细胞级分三次,
  8. 使用流式细胞仪通过流式细胞术分析评估各种细胞级分的纯度。用异硫氰酸荧光素(FITC)结合的抗CD3(检测总T细胞的克隆145-2C11),藻红蛋白(PE)结合的抗CD19(克隆1D3以检测B细胞),FITC-结合的抗CD19- CD4(克隆RM4-5以检测CD4 + T细胞)和PE缀合的抗CD8(克隆53-6.7以检测CD8 + T细胞),并分析对于阳性细胞,按照标准程序

食谱

  1. 分离缓冲区
    1×具有5mM EDTA和0.5%BSA的PBS

致谢

作者感谢德国研究基金会(DFG)和大学埃尔兰根 - 纽伦堡资助。 我们感谢A. von Berg和L. Sologub出色的技术援助。

参考文献

  1. Miltenyi,S.,Muller,W.,Weichel,W。和Radbruch,A。(1990)。 使用MACS的高梯度磁性细胞分离 Cytometry 11 (2):231-238
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Copyright: © 2013 The Authors; exclusive licensee Bio-protocol LLC.
引用:Weigmann, B. (2013). Cell Isolation of Spleen Mononuclear Cells. Bio-protocol 3(9): e689. DOI: 10.21769/BioProtoc.689.
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