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Assessing Mitochondrial Transport via Cytoplasmic Nanotubular Bridges in Cells
通过细胞中细胞质纳米管桥接评估线粒体的转运   

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Abstract

This protocol aims to study intercellular transport of mitochondria, dynamic cellular organelles via tunnelling nanotubes (TNT), a cell membrane extension of cytoskeletal elements. The nanotubular bridges or the tunnelling nanotube highways are one of the emerging new cell-to-cell communication systems which mediates exchange of cellular materials, most importantly as in our observation, mitochondria. Mesenchymal stem cells (MSC) have been well studied to be endowed with a highly efficient intercellular mitochondrial donation ability and this property is now proven crucial to its functional role of rescue in cellular therapy.

Keywords: Mitochondrial transfer(线粒体移植), Nanotube(纳米管), Intercellular transport(胞间运输), Miro1(miro1)

Materials and Reagents

  1. Cells
    Mesenchymal stem cells (Human or Mouse derived in passage 3-7), any fibroblasts cell lines (HSMC, 3T3) or primary cells. Even epithelial [BEAS-2b (ATCC, catalog number: CRL-9609 ), NHBE (Lonza, catalog number: CC-2540 ), A549, LA-4, MLE- 12] and endothelial cells (HUVEC) also show such capacity but at comparatively minimal efficiency.

  2. Reagents
    1. CellLight® Mitochondria-GFP, BacMam 2.0 (Life Technologies, catalog number: C10600 )
    2. CellLight® Mitochondria-RFP, BacMam 2.0 (Life Technologies, catalog number: C10505 )
    3. MitoTracker® Green FM (Life Technologies, catalog number: M7514 )
    4. MitoTracker® Deep Red FM (Life Technologies, catalog number: M22426 )
    5. Alexa Fluor® 594 phalloidin (Life Technologies, catalog number: A12381 ) or fluorescein phalloidin (Life Technologies, catalog number: F432 )
    6. Alexa Fluor® 488 phalloidin (Life Technologies, catalog number: A12379 )
    7. 2% paraformaldehyde (SigmaAldrich, catalog number: P6148 ) diluted in 1x PBS
    8. ProLong® Gold Antifade Mountant with DAPI (Life Technologies, catalog number: P36931 )
    9. Respective cell culture media
      1. DMEM (Sigma-Aldrich, catalog number: D5523 ) + 10% FBS for Human MSC (HuMSC), Fibroblasts 3T3, A549
      2. Mouse MesenCult™ (STEMCELL Technologies, catalog number: 05512 ) for mouse MSC
      3. Ham’s-F 12 (Sigma-Aldrich, catalog number: N6760 ) + 15% FBS for LA-4, MLE- 12
      4. BEGM (Lonza, catalog number: CC3170 ) for NHBE and BEAS-2b
      5. M-199 (Sigma-Aldrich, catalog number: M2520 ) + 15% FBS (Life Technologies, InvitrogenTM, catalog number: 10270 ) for HUVECs (primary cultured)
    10. TritonX-100 (Sigma-Aldrich, catalog number: T9284 )
    11. 1x phosphate buffer saline (PBS)

Equipment

  1. Lab-tek II Chambered Coverglass w/cover #1.5 Borosilicate Sterile (Sigma-Aldrich)
  2. 75 x 25 mm microscope slides (J. Melvin Freed Brand)
  3. 22 x 22 Esco microscope square cover glass with no. 1 thickness (Erie Scientific Company)
  4. Live cell fluorescent microscope (Leica Microsystems, model: DMI6000 )
  5. Confocal microscope (Leica Microsystems, model: TCSSP5 )
  6. BD FACS Aria (BD Biosciences)
  7. BD FACS Calibur (BD Biosciences)

Procedure

  1. Sample preparation
    1. The typical cells (e.g. Human MSC or HuMSC and BEAS-2b) must first be grown separately and differentially transfected (with 20 µl volume for approx 30,000 cells seeded) either of CellLight® Mitochondria-GFP, BacMam 2.0 and CellLight® Mitochondria-RFP, BacMam 2.0. After 24 h, the respective cells are then co-cultured. Alternatively they can also be stained with 2 μl/ml (of cell growth media) from 100 μM stock solution of Mitotracker dyes for 10-20 min in 37 °C before the coculture (e.g. HuMSC with MitoTracker® DeepRed and BEAS-2b with MitoTracker® Green).
    2. Post dye-incubation, the cell media containing the dye is washed using 1x PBS and fresh cell growth media is added.
    3. For live cell imaging of monoculture population, the cells stained either of the mitotracker dye can also be stained with phalloidin (F432, Invitrogen or A12381, Invitrogen) 1:250 in PBS to view the actin constituted TNT.
    4. The cells have to be seeded in 2- or 4-chambered slides for live cell image acquisition (about 10-30,000 cells in each well). In case of co-cultured cells, respective growth media has to be added in equal volumes. The two types of cells are seeded in equal ratio and post 3-4 h of co-culture, mitochondrial donation can be observed.
    5.  For fixed cell immunocytochemistry, the stained cells have to be reseeded onto cover glasses. Wash and wipe cover glass with 70% ethanol and place them in each well of a 6-well plate. Seed 30,000 cells in each well for monocultures or equi-ratio of co-cultures and then can proceed on with the staining. The cells have to be fixed in 2% paraformaldehyde (in 1x PBS) for about 20 min at RT or overnight at 4 °C and phalloidin staining (20 min incubation) can be done post permeabilization (permeabilization buffer: 0.1% TritonX-100 in PBS for 15 min) . The cover glass with the cells have to be finally mounted on glass slides with ProLong® Gold Antifade Mountant with DAPI. The edges of the cover glass are sealed with nail enamel.
    6. For FACS quantification of mitochondria transported, the differentially stained co-cultured cells have to be trypsinized (0.125% Trypsin+ 0.01 M EDTA) and cell pellet obtained has to be diluted in PBS or FACS buffer for acquiring.

  2. Flurimetric quantification
    The FACS Calibur/ FACS Aria was used to acquire a quantitative estimate of mitochondria transported by MSC. The count of mitochondria (as labeled in MSC) transported into epithelial or other specific gated population of cells is thus determined.

  3. Image acquisition
    The image acquisitions can be done with either 63x or 100x objective and TNT can be imaged with DIC view. The various dyes and respective lasers with excitation and emission range are tabulated as follows:

    Dyes/Probes
    Lasers
    Excitation
    Emission
    MitoGFP
    ARGON
    488 nm
    500-540 nm
    MitoRFP
    DPSS 561
    584 nm
    580-620 nm
    Mitotracker green
    ARGON
    488 nm
    500-540 nm
    Mitotracker red
    DPSS 561
    575 nm
    580-630 nm

Recipes

All reagents, probes and dyes were re-constituted in and as mentioned in procedures or as per manufacturer’s protocol.

Acknowledgments

This research work was supported by grants MLP 5502 and BSC 0403 from the Council of Scientific and Industrial Research, Govt. of India.

References

  1. Ahmad, T., Aggarwal, K., Pattnaik, B., Mukherjee, S., Sethi, T., Tiwari, B. K., Kumar, M., Micheal, A., Mabalirajan, U., Ghosh, B., Sinha Roy, S. and Agrawal, A. (2013). Computational classification of mitochondrial shapes reflects stress and redox state. Cell Death Dis 4: e461.
  2. Ahmad, T., Mukherjee, S., Pattnaik, B., Kumar, M., Singh, S., Kumar, M., Rehman, R., Tiwari, B. K., Jha, K. A., Barhanpurkar, A. P., Wani, M. R., Roy, S. S., Mabalirajan, U., Ghosh, B. and Agrawal, A. (2014). Miro1 regulates intercellular mitochondrial transport & enhances mesenchymal stem cell rescue efficacy. EMBO J 33(9): 994-1010.

简介

此协议旨在研究线粒体,动态细胞器通过隧道纳米管(TNT),细胞骨架元素的细胞膜扩展的细胞间运输。 纳米管桥或隧道纳米管高速公路是新兴的新的细胞到细胞通信系统之一,其介导细胞材料的交换,最重要的是在我们的观察中,线粒体。 间充质干细胞(MSC)已经被充分研究以赋予高效的细胞间线粒体捐赠能力,并且这种性质现在被证明对于其在细胞治疗中的挽救的功能作用是至关重要的。

关键字:线粒体移植, 纳米管, 胞间运输, miro1

材料和试剂

  1. 单元格
    间充质干细胞(来自3-7代的人或小鼠),任何成纤维细胞细胞系(HSMC,3T3)或原代细胞。 甚至上皮[BEAS-2b(ATCC,目录号:CRL-9609),NHBE(Lonza,目录号:CC-2540),A549,LA-4,MLE-12]和内皮细胞(HUVEC) 效率相对较低。

  2. 试剂
    1. CellLight Mitochondria-GFP,BacMam 2.0(Life Technologies,目录号:C10600)
    2. CellLight Mitochondria-RFP,BacMam 2.0(Life Technologies,目录号:C10505)
    3. MitoTracker ® Green FM(Life Technologies,目录号:M7514)
    4. MitoTracker 深红色FM(Life Technologies,目录号:M22426)
    5. Alexa Fluor?594鬼笔环肽(Life Technologies,目录号:A12381)或荧光素鬼笔环肽(Life Technologies,目录号:F432)
    6. Alexa Fluor 48鬼笔环肽(Life Technologies,目录号:A12379)
    7. 用1×PBS稀释的2%多聚甲醛(SigmaAldrich,目录号:P6148)
    8. ProLong ®带有DAPI的Gold Antifade Mountant(Life Technologies,目录号:P36931)
    9. 相应的细胞培养基
      1. 用于人MSC(HuMSC),成纤维细胞3T3,A549的DMEM(Sigma-Aldrich,目录号:D5523)+ 10%FBS
      2. 用于小鼠MSC的小鼠MesenCult TM(STEMCELL Technologies,目录号:05512)
      3. Ham's-F12(Sigma-Aldrich,目录号:N6760)+ LA-4,MLE-12的15%FBS
      4. 用于NHBE和BEAS-2b的BEGM(Lonza,目录号:CC3170)
      5. M-199(Sigma-Aldrich,目录号:M2520)+ 15%FBS(Life Technologies,Invitrogen TM ,目录号:10270) 培养)
    10. TritonX-100(Sigma-Aldrich,目录号:T9284)
    11. 1×磷酸盐缓冲盐水(PBS)

设备

  1. Lab-tek II Chambered盖玻片w /盖#1.5硼硅酸盐无菌(Sigma-Aldrich)
  2. 75×25mm显微镜载玻片(J.Melvin Freed Brand)
  3. 22 x 22 Esco显微镜方形封面玻璃没有。 1厚度(Erie Scientific Company)
  4. 活细胞荧光显微镜(Leica Microsystems,型号:DMI6000)
  5. 共焦显微镜(Leica Microsystems,型号:TCSSP5)
  6. BD FACS Aria(BD Biosciences)
  7. BD FACS Calibur(BD Biosciences)

程序

  1. 样品准备
    1. 典型的小区(例如人类MSC或HuMSC和BEAS-2b)必须首先是   分别生长和差异转染(用20μl体积 约30000个细胞接种)或CellLight线粒体-GFP, BacMam 2.0和CellLight Mitochondria-RFP,BacMam 2.0。 24小时后, 然后共培养各自的细胞。 或者它们也可以 用来自100μM储备液的2μl/ml(细胞生长培养基)染色 的Mitotracker染料在37℃下孵育10-20分钟(例如 HuMSC与MitoTracker DeepRed和BEAS-2b与MitoTracker 绿色)。
    2. 染料后孵育,使用1×PBS洗涤含有染料的细胞培养基,并加入新鲜的细胞生长培养基。
    3. 对于单细胞群体的活细胞成像,细胞染色 任何一种mitotracker染料也可以用鬼笔环肽(F432,  Invitrogen或A12381,Invitrogen)在PBS中1:250以观察肌动蛋白 构成TNT
    4. 细胞必须接种在2-或4-室中 载玻片用于活细胞图像采集(每个约10-30,000个细胞) 好)。在共培养细胞的情况下,必须有相应的生长培养基 以等体积加入。两种类型的细胞以相等的比例接种  和3-4小时的共培养,可观察到线粒体捐赠
    5.  对于固定细胞免疫细胞化学,染色的细胞必须 重新接种到盖玻片上。用70%乙醇洗涤并擦拭玻璃盖 并将其置于6孔板的每个孔中。种子30,000个细胞  用于单培养或等比例的共培养,然后可以进行  染色。细胞必须在2%多聚甲醛中固定  (在1x PBS中)在室温下约20分钟或在4℃下过夜和鬼笔环肽 可以在透化后(透化缓冲液:0.1%TritonX-100的PBS溶液,15分钟)进行染色(20分钟孵育)。 封面   玻璃与细胞必须最终安装在玻璃载玻片上 ProLong ®金防褪色剂。 盖玻璃的边缘 用指甲油密封。
    6. 对于FACS定量 线粒体转运,差异染色的共培养细胞 必须用胰蛋白酶处理(0.125%胰蛋白酶+ 0.01M EDTA)和细胞沉淀 必须在PBS或FACS缓冲液中稀释获得。

  2. 比色量化
    FACS Calibur/FACS Aria用于获得由MSC转运的线粒体的定量估计。 因此确定转运到上皮或其他特定门控细胞群体中的线粒体计数(如在MSC中标记的)。

  3. 图像采集
    图像采集可以用63x或100x物镜完成,TNT可以用DIC视图成像。 各种染料和具有激发和发射范围的相应激光器列表如下:

    染料/探针
    激光
    励磁
    发光
    MitoGFP
    ARGON
    488 nm
    500-540 nm
    MitoRFP
    DPSS 561
    584 nm
    580-620 nm
    Mitotracker绿色
    ARGON
    488 nm
    500-540 nm
    Mitotracker红色
    DPSS 561
    575 nm
    580-630 nm

食谱

所有试剂,探针和染料在过程中或如制造商的方案中所述重组。

致谢

该研究工作得到了来自科学和工业研究理事会(Government of Scientific and Industrial Research,Govt。)的MLP 5502和BSC 0403授权的支持。 的印度。

参考文献

  1. Ahmad,T.,Aggarwal,K.,Pattnaik,B.,Mukherjee,S.,Sethi,T.,Tiwari,BK,Kumar,M.,Micheal,A.,Mabalirajan,U.,Ghosh, Roy,S。和Agrawal,A。(2013)。 线粒体形状的计算分类反映了应激和氧化还原状态。细胞死亡 4:e461。
  2. Ahmad,T.,Mukherjee,S.,Pattnaik,B.,Kumar,M.,Singh,S.,Kumar,M.,Rehman,R.,Tiwari,BK,Jha,KA,Barhanpurkar,AP,Wani, ,Roy,SS,Mabalirajan,U.,Ghosh,B。和Agrawal,A。(2014)。 Miro1调节细胞间线粒体转运& 增强间充质干细胞拯救功效。 EMBO J 33(9):994-1010。
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免责声明 × 为了向广大用户提供经翻译的内容,www.bio-protocol.org 采用人工翻译与计算机翻译结合的技术翻译了本文章。基于计算机的翻译质量再高,也不及 100% 的人工翻译的质量。为此,我们始终建议用户参考原始英文版本。 Bio-protocol., LLC对翻译版本的准确性不承担任何责任。
Copyright: © 2015 The Authors; exclusive licensee Bio-protocol LLC.
引用: Readers should cite both the Bio-protocol article and the original research article where this protocol was used:
  1. Mukherjee, S., Ahmad, T. and Agrawal, A. (2015). Assessing Mitochondrial Transport via Cytoplasmic Nanotubular Bridges in Cells. Bio-protocol 5(15): e1542. DOI: 10.21769/BioProtoc.1542.
  2. Ahmad, T., Mukherjee, S., Pattnaik, B., Kumar, M., Singh, S., Kumar, M., Rehman, R., Tiwari, B. K., Jha, K. A., Barhanpurkar, A. P., Wani, M. R., Roy, S. S., Mabalirajan, U., Ghosh, B. and Agrawal, A. (2014). Miro1 regulates intercellular mitochondrial transport & enhances mesenchymal stem cell rescue efficacy. EMBO J 33(9): 994-1010.
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