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Purification and Fluorescent Labeling of Exosomes
外泌体的纯化和荧光标记   

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Abstract

Exosomes are small membrane vesicles of endocytic origin secreted into the extracellular environment from a variety of different cells, and are thought to play important roles in intercellular communications. Here, we provide a useful protocol to purify the exosomes released from cell lines using sucrose gradient centrifugation. In this protocol, we also applied a red-fluorescent lipophilic dye, DiI, which is incorporated in the outer membrane of exosomes. This fluorescently labeled exosomes allow us to visualize individual exosomes by a confocal laser scanning microscope.

Keywords: Exosome(外切体), Extracellular vesicle(胞囊), Fluorescent labeling(荧光标记), Microscope(显微镜)

Materials and Reagents

  1. Burkitt’s Lymphoma B cell lines (e.g. Mutu-, Mutu I, Mutu III cell lines)
  2. RPMI 1640 medium (Wako Chemicals USA, catalog number: 189-02025 )
  3. Sucrose (Sigma-Aldrich, catalog number: S7903 )
  4. Anti-CD63 monoclonal antibody (clone MEM-250) (Abnova, catalog number: MAB0931 )
  5. Bradford protein assay kit (Bio-Rad Laboratories, catalog number: 500-0006JA )
  6. 1, 1'-dioctadecyl-3, 3, 3', 3'-tetramethylindocarbocyanine perchlorate (DiI) (Life Technologies, catalog number: D3911 )
  7. Fetal Bovine Serum (FBS) (Sigma-Aldrich, catalog number: F9423 )
  8. Tris
  9. NaCl
  10. EDTA
  11. Exosome-depleted FBS (see Recipes)
  12. TNE buffer (see Recipes)
  13. 0.25-2.5 M sucrose gradient in TNE buffer (see Recipes)

Equipment

  1. 10 cm dish
  2. Centrifuge (Eppendorf, model: 5810R or that with equivalent equipment spec)
  3. Ultracentrifuge (Beckman Coulter, model: Optima L-80 XP or that with equivalent equipment spec)
  4. 37 °C, 5% CO2 cell culture incubator
  5. Autopipette
  6. 50 ml polypropylene concal plastic tubes (BD Biosciences, Falcon®, catalog number: 352070 or that with equivalent spec)
  7. SW28 rotor (Beckman Coulter, model: 342204 )
  8. SW40Ti rotor (Beckman Coulter, model: 331301 )
  9. Polyallomer centrifuge tubes 1 x 3½ in (25 x 89 mm) for SW28 rotor (Beckman Coulter, catalog number: 326823 )
  10. Polyallomer centrifuge tubes 9/16 x 3½ in (14 x 89 mm) for SW41Ti rotor (Beckman Coulter, catalog number: 331372 )
  11. Spectrometer
  12. Fluorescent or confocal laser scanning microscope

Procedure

  1. Purification of exosomes
    1. Burkitt’s Lymphoma cell lines are grown up from 1 x 107 (in one 10 cm dish) to 2 x 108 cells (in twenty 10 cm dishes) in 200 ml RPMI 1640 medium containing 10% exosome-depleted FBS in the 5% CO2 incubator at 37 °C.
    2. Culture medium containing exosomes are harvested and centrifuged in 50 ml conical tubes at 1,500 rpm for 10 min at room temperature to remove cells.
    3. The supernatant is centrifuged in 50 ml conical tubes at 3,500 rpm for 15 min at room temperature to remove cell debris.
    4. The supernatant is ultracentrifuged in polyallomer centrifuge tubes at 25,000 rpm for 1 h at 4 °C with an SW28 rotor.
    5. The pelleted exosomes are resuspended in 100 µl TNE buffer over night at 4 °C.
    6. The exosomes are fractionated by use of a 0.25-2.5 M sucrose gradient in TNE buffer in polyallomer centrifuge tubes at 25,000 rpm for 4 h at 4 °C with an SW40Ti rotor. After that, you will see a band derived from exosomes (if you collect 1 ml of each fraction from the top, exosome fraction usually locates around 6th fraction from the top).
    7. The band is collected (about 1 ml) carefully with an autopipette.
    8. Fractionated exosomes are ultracentrifuged at 25,000 rpm for 1 h at 4 °C with an SW40Ti rotor.
    9. The pelleted exosomes is resuspended in 100 ~ 200 µl TNE buffer over night at 4 °C.
    10. The total protein concentration in the fractions is determined by the Bradford protein assay.
    11. The fraction containing exosomes (4 µg, each) is confirmed by western blot analysis with anti-CD63 monoclonal antibody (1:1,000 dilution) (Figure 1).


      Figure 1. Purified exosomes derived from Burkitt’s lymphoma Mutu cell lines. Exosomes were purified from culture medium of Mutu- (1st lane), Mutu I (2nd lane), and Mutu III (3rd lane) cells. 4 µg of exosomes were analyzed by western blot with anti-CD63. The arrow indicates the bands that correspond to CD63.

  2. Fluorecent labeling of exosomes
    1. 1 ml of fractionated exosomes (100 ng/ml) is incubated with 6 µl of 10 µM stock solution of 1, 1'-Dioctadecyl-3, 3, 3', 3'-Tetramethylindocarbocyanine Perchlorate (DiI) in methanol for 1 h in the dark at room temperature with gentle agitation.
    2. Confirm the efficiency of labeling with small amount of exosomes under fluorescent or confocal laser scanning microscope.
    3. Aliquot, stored at -80 °C.

Recipes

  1. Exosome-depleted FBS
    Ultracentrifuge FBS at 25,000 rpm for 4 h at 4 °C
    Collect supernatant and stored at 4 °C
  2. TNE buffer
    10 mM Tris-HCl (pH 7.6)
    100 mM NaCl
    1 mM EDTA
    Stored at room temperature
  3. 0.25-2.5 M sucrose gradient in TNE buffer
    Prepare 0.25 M and 2.5 M sucrose in TNE buffer (Figure 2)
    1. Fill 2.5 M sucrose solution up to the half the height of polyallomer centrifuge tubes (approximately 6 ml)
    2. Add 0.25 M sucrose solution in layers up to the top of tubes (approximately 6 ml) and plug the tubes with rubber plugs
    3. Lay down the tubes with the top slightly higher than bottom (use thin tube floater as a pillow) for al leaset 1.5 h at room temperature
    4. Stand the tubes slowly and keep at 4 °C until just before use (storable up to 2 days)


    Figure 2. Preparation of 0.25-2.5 M sucrose gradient in TNE buffer. (a) Fill 2.5 M sucrose solution up to the half the height of polyallomer centrifuge tubes (approximately 6 ml). (b) Add 0.25 M sucrose solution in layers up to the top of tubes (approximately 6 ml) and plug the tubes with rubber plugs. (c) Lay down the tubes with the top slightly higher than bottom (use thin tube floater as a pillow) for at least 1.5 h at room temperature. (d) Stand the tubes slowly and keep at 4 °C until just before use (storable up to 2 days).

Acknowledgments

This protocol has been adapted from a previously published paper (Nanbo et al., 2013). This work was supported in part by Grant for Funding from Basic Science research projects from The Sumitomo Foundation; Akiyama Life Science Foundation; Grant-in-Aid for Scientific Research; Shiseido Female Researcher Science Grant; The Sagawa Foundation for Promotion of Cancer Research.

References

  1. Nanbo, A., Kawanishi, E., Yoshida, R. and Yoshiyama, H. (2013). Exosomes derived from Epstein-Barr virus-infected cells are internalized via caveola-dependent endocytosis and promote phenotypic modulation in target cells. J Virol 87(18): 10334-10347.
  2. Nanbo, A., Imai, M., Watanabe, S., Noda, T., Takahashi, K., Neumann, G., Halfmann, P. and Kawaoka, Y. (2010). Ebolavirus is internalized into host cells via macropinocytosis in a viral glycoprotein-dependent manner. PLoS Pathog 6(9): e1001121.

简介

外来体是从各种不同细胞分泌到细胞外环境中的内吞起源的小膜囊泡,并且被认为在细胞间通讯中起重要作用。 在这里,我们提供了一个有用的协议,以纯化使用蔗糖梯度离心从细胞系释放的外来体。 在这个协议,我们还应用红色荧光亲脂染料DiI,其被并入外来体的外膜。 这种荧光标记的外来体允许我们通过共聚焦激光扫描显微镜可视化单个外来体

关键字:外切体, 胞囊, 荧光标记, 显微镜

材料和试剂

  1. 伯基特氏淋巴瘤B细胞系(例如 Mutu - ,Mutu I,Mutu III细胞系)
  2. RPMI 1640培养基(Wako Chemicals USA,目录号:189-02025)
  3. 蔗糖(Sigma-Aldrich,目录号:S7903)
  4. 抗CD63单克隆抗体(克隆MEM-250)(Abnova,目录号:MAB0931)
  5. Bradford蛋白测定试剂盒(Bio-Rad Laboratories,目录号:500-0006JA)
  6. 1,1'-二(十八烷基)-3,3,3',3'-四甲基吲哚羰花青高氯酸盐(DiI)(Life Technologies,目录号:D3911)
  7. 胎牛血清(FBS)(Sigma-Aldrich,目录号:F9423)
  8. Tris
  9. NaCl
  10. EDTA
  11. 外来体耗尽的FBS(参见配方)
  12. TNE缓冲区(参见配方)
  13. 0.25-2.5M蔗糖梯度在TNE缓冲液(见配方)中

设备

  1. 10厘米培养皿
  2. 离心机(Eppendorf,型号:5810R或同等设备规格)
  3. 超速离心机(Beckman Coulter,型号:Optima L-80 XP或具有同等设备规格)
  4. 37℃,5%CO 2细胞培养箱中培养
  5. 自动移液器
  6. 50ml聚丙烯锥形塑料管(BD Biosciences,Falcon ,目录号:352070或具有同等规格的)
  7. SW28转子(Beckman Coulter,型号:342204)
  8. SW40Ti转子(Beckman Coulter,型号:331301)
  9. 用于SW28转子(Beckman Coulter,目录号:326823)的聚合物离心管1×3/2英寸(25×89mm)
  10. 用于SW41Ti转子(Beckman Coulter,目录号:331372)的聚合物离心管9/16×3/2英寸(14×89mm)
  11. 光谱仪
  12. 荧光或共焦激光扫描显微镜

程序

  1. 外来体的纯化
    1. 伯基特氏淋巴瘤细胞系在200ml RPMI中从1×10 7个(在一个10cm培养皿中)培养至2×10 8个细胞(在二十个10cm培养皿中) 1640培养基在含有10%外来体缺失的FBS的培养基中在37℃下在5%CO 2培养箱中培养。
    2. 文化   收获含有外来体的培养基,并在50ml中离心 锥形管在室温下以1,500rpm离心10分钟以除去 细胞。
    3. 将上清液在50ml锥形管中在室温下以3500rpm离心15分钟以除去细胞碎片
    4. 将上清液在多聚体离心管中在25,000rpm下在4℃下用SW28转子超速离心1小时。
    5. 将沉淀的外来体在4℃下在100μlTNE缓冲液中重悬浮过夜
    6. 的   外泌体通过使用0.25-2.5M蔗糖梯度 TNE缓冲液在多聚集体离心管中在4℃下以25,000rpm离心4小时   与SW40Ti转子。 之后,你会看到一个乐队派生自 外来体(如果你从顶部收集每个级分1毫升,外来体 馏分通常位于从顶部起约6个馏分)。
    7. 用自动吸移管小心收集带(约1ml)。
    8. 将分级外核体用SW40Ti转子在25,000rpm,4℃下超速离心1小时。
    9. 将沉淀的外来体在4℃下在100〜200μlTNE缓冲液中重悬浮过夜
    10. 通过Bradford蛋白质测定确定级分中的总蛋白质浓度
    11. 的 通过蛋白质印迹证实含有外来体的部分(4μg,每个) 用抗CD63单克隆抗体(1:1000稀释)分析(图 1)。


      图1.源自伯基特氏淋巴瘤Mutu细胞系的纯化的外泌体。从Mutu细胞(第一泳道)的培养基中纯化外来体,Mutu 一世 (第2泳道)和Mutu III(第3泳道)细胞。 4μg外来体 通过用抗CD63的western印迹分析。 箭头表示乐队 对应于CD63。

  2. 荧光标记外来体
    1. 1   ml的分级外来体(100ng/ml)与6μl的10μM温育 1,1'-二(十八烷基)-3,3,3',6' 3'-四甲基吲哚羰花青高氯酸盐(DiI)在甲醇中1小时 在室温下黑暗,轻轻搅拌
    2. 在荧光或共焦激光扫描显微镜下确认用少量外来体标记的效率
    3. 等分试样,储存于-80℃

食谱

  1. 外来体耗尽的FBS
    超速离心机FBS在25,000rpm,4℃,4小时 收集上清液并在4℃下保存
  2. TNE缓冲区
    10mM Tris-HCl(pH7.6) 100 mM NaCl
    1mM EDTA
    在室温下贮存
  3. 0.25-2.5M蔗糖梯度在TNE缓冲液中 在TNE缓冲液(图2)中制备0.25M和2.5M蔗糖
    1. 将2.5M蔗糖溶液填充至多聚离心管的高度的一半(约6ml)
    2. 在层中加入0.25M蔗糖溶液至管顶部(约6ml),并用橡皮塞
      塞住试管
    3. 放置管的顶部稍高于底部(使用薄 管浮子作为枕头)用于室温1.5小时
    4. 慢慢地放置管,并保持在4°C,直到使用前(可储存达2天)


    图2.在TNE缓冲液中制备0.25-2.5M蔗糖梯度。(a)将2.5M蔗糖溶液填充至高度的一半 多聚集体离心管(约6ml)。 (b)加入0.25M 蔗糖溶液,直到管的顶部(约6ml) 并用橡胶塞塞住管子。 (c)放下管子 顶部略高于底部(使用细管浮子作为枕头)为 在室温下至少1.5小时。 (d)慢慢站立管并保持 在4°C,直到就在使用前(可储存达2天)

致谢

该协议已经从先前发表的文章(Nanbo等人,2013年)改编。 这项工作得到了住友基金会的基础科学研究项目资金拨款的支持; 秋山生命科学基金会; 科学研究助学金; 资生堂女研究员科学资助; 佐川促进癌症研究基金会。

参考文献

  1. Nanbo,A.,Kawanishi,E.,Yoshida,R。和Yoshiyama,H。(2013)。 外来体   来源于Epstein-Barr病毒感染的细胞 caveola依赖内吞和促进表型调节 靶细胞。 Virol 87(18):10334-10347。
  2. Nanbo,A.,Imai,M.,Watanabe,S.,Noda,T.,Takahashi,K.,Neumann,G.,Halfmann,P.and Kawaoka,Y。 埃博拉病毒通过内部化到宿主细胞中 > macropinocytosis in a viral glycoprotein-dependent manner。 PLoS Pathog 6(9):e1001121。
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Copyright: © 2014 The Authors; exclusive licensee Bio-protocol LLC.
引用:Nanbo, A., Kawanishi, E., Yoshida, R. and Yoshiyama, H. (2014). Purification and Fluorescent Labeling of Exosomes. Bio-protocol 4(8): e1103. DOI: 10.21769/BioProtoc.1103.
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