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Mitochondrial Transmembrane Potential (ψm) Assay Using TMRM
采用TMRM进行线粒体膜电位(ψm)分析   

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Abstract

During cellular respiration, nutrients are oxidized to generate energy through a mechanism called oxidative phosphorylation, which occurs in the mitochondria. During oxidative phosphorylation, the gradual degradation of molecules through the TCA cycle releases electrons from the covalent bonds that are broken. These electrons are captured by NAD+ through its reduction into NADH. Finally, NADH transports the electrons to the complexes of the electron chain in the internal membrane of mitochondria. These complexes use the energy released by the electrons to pump protons into the intermembrane space, generating an electrochemical gradient across the internal membrane of mitochondria, which provides energy for the ATP-synthase complex, ultimately producing adenosine triphosphate (ATP). We assessed the mitochondrial membrane potential (ψm) using tetramethylrhodamine methyl ester (TMRM), a cell-permeant, cationic, red fluorescent dye. To measure specifically the mitochondrial membrane potential (ψm) we quantified the fluorescence intensity before and after applying FCCP, a mitochondrial electron chain uncoupler. The difference of intensity before and after applying FCCP corresponds specifically to the mitochondrial membrane potential. We analyzed mitochondrial membrane potential (ψm) by cytofluorimetry. The ratio between the total level of signal and the signal generated after uncoupling provided a normalized value for the difference in cell size. Furthermore, to normalize for the different size of cells that we were analyzing we have analyzed TMRM in live imaging using IN Cell Analyzer, so that the level of mitochondrial membrane potential could be detected per unit of mitochondrial membrane area measured. Thus, our protocol can also be used to compare the mitochondrial membrane potential of cells that are different in size.

Materials and Reagents

  1. Murine Embrionic Fbroblasts (MEF)
  2. Phenol-red free HBSS (Gibco®, catalog number: 14175-079 )
  3. 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) (Gibco®, catalog number: 15630-080 )
  4. Tetramethylrhodamine methyl ester (TMRM) (Life Technologies, catalog number: T668 )
  5. Cyclosporin H (Enzo Life Sciences, catalog number: ALX-380-286-M001 )
  6. Hoechst 33342
  7. Carbonylcyanide-p-trifluoromethoxyphenyl hydrazone FCCP (Sigma-Aldrich, catalog number: C2920-10MG )
  8. Trypsin (451) - Trypsin, 0.05% (1X) with 0.53 mM EDTA 4Na, liquid, 20 x 100 ml
    (Gibco®, catalog number: 25300096 )

Equipment

  1. IN Cell Analyzer 1000 (General Electric Company)
  2. 37 °C 5% CO2 Cell culture incubator
  3. 12 well pates (Corning, Costar®, catalog number: CLS 3513 )
  4. FACScan (BD)
  5. Centrifuge spinning speed: 15,682,186 x g (G-force) (13,000 rpm, 8,3 cm radius)

Software

  1. IN Cell Investigator Analysis software (General Electric Company)
  2. BD CellQuest software
  3. FlowJo software

Procedure

  1. For quantitative real-time analysis
    1. Cells are plated at 50% cell confluence.
    2. Cells are incubated for 30 min at 37 °C in phenol-red free HBSS with 10 mM HEPES, 20 nM TMRM, 2 μM cyclosporine-H, inhibitor of multidrug resistance pump activity but not of the permeability transition pore (multi drug resistance pump activity can affect the mitochondrial lloading with TMRM) and 2 μg/ml Hoechst 33342, nuclear dye that can be used in living cells.
    3. Sequential images were taken before (3 time points) and after 4 μM FCCP (3 time points) was injected in a motorized way for TMRM (535 nm excitation filter; 600 nm emission) and Hoechst 33342 (360 nm excitation filter; 460 nm emission) in different fields (4 fields acquired for each well and 6 wells for each experimental condition) every 3 min with an IN CELL Analyzer 1000.
    4. Images are automatically analyzed with IN Cell Investigator Analysis software to measure the TMRM intensity. Fluorescence intensity is measured on the average of 10 points randomly selected for each field at the first and the last time point, the background corresponding to an area without cell is removed for each field.
    5. The fluorescence intensity measured at the last time point (after applying FCCP) is normalized by the fluorescence intensity measured at the first time point of the experiment.

  2. For FACS analysis
    1. Cells are plated at 50% confluence.
    2. Cells are trypsinized (at least 2 millions of cells).
    3. Cells are centrifuged for 5 min at G-force 13,62.
    4. Cells are washed in PBS, resuspended in phenol-red free HBSS with 10 mM HEPES and counted.
    5. Cells (300,000 cells per tube, each condition done in triplicate) are resuspended the in phenol-red free HBSS with 10 mM HEPES and 20 nM TMRM in the presence of 2 μM of the multidrug resistance pump inhibitor cyclosporine-H and incubated for 30 min at 37 °C. In parallel, cells are incubated 5 min in the same conditions with an uncoupling agent, 4 μM FCCP, to measure the specific mitochondria staining.
    6. FACS analysis for TMRM was performed on BD FACScan with CellQuest software and analysed using FlowJo software. Fluorescence intensity of TMRM was calculated by gating on live cells.
    7. The intensity of the fluorescence with FCCP was normalized by the intensity without FCCP in order to rule out the possibility that the difference of the intensity is only or mainly caused by the cell sizes.

Acknowledgments

We thank all the co-authors of the article: Chiaravalli, M., Mannella, V., Ulisse, V., Quilici, G., Pema, M., Song, X. W., Xu, H.,  Mari, S., Qian, F., Pei, Y. and Musco, G., the other members of the lab Boletta, Casari, G. and Cassina, L. and the San Raffaele microscopy facility (Alembic).

References

  1. Rowe, I., Chiaravalli, M., Mannella, V., Ulisse, V., Quilici, G., Pema, M., Song, X. W., Xu, H., Mari, S., Qian, F., Pei, Y., Musco, G. and Boletta, A. (2013). Defective glucose metabolism in polycystic kidney disease identifies a new therapeutic strategy. Nat Med 19(4): 488-493.

简介

在细胞呼吸期间,营养物通过称为氧化磷酸化的机制被氧化以产生能量,所述机制发生在线粒体中。在氧化磷酸化过程中,分子通过TCA循环的逐渐降解从被破坏的共价键释放电子。这些电子被NAD +捕获,通过其还原成NADH。最后,NADH将电子传输到线粒体内膜中的电子链的复合物。这些复合物使用由电子释放的能量将质子泵入膜间空间,产生穿过线粒体内膜的电化学梯度,其为ATP-合酶复合物提供能量,最终产生三磷酸腺苷(ATP)。我们使用四甲基罗丹明甲酯(TMRM),一种细胞渗透性,阳离子,红色荧光染料评估线粒体膜电位(ψsub)。为了特异性地测量线粒体膜电位(ψm),我们定量了应用FCCP(线粒体电子链解偶联)之前和之后的荧光强度。应用FCCP之前和之后的强度差异具体对应于线粒体膜电位。我们通过细胞荧光法分析线粒体膜电位(ψm)。信号的总电平与解耦之后产生的信号之间的比率提供了对于单元大小差异的归一化值。此外,为了归一化我们分析的不同大小的细胞,我们已经使用IN细胞分析仪分析了活体成像中的TMRM,使得可以测量每单位线粒体膜面积的线粒体膜电位水平。因此,我们的协议也可以用于比较不同大小的细胞的线粒体膜电位。

材料和试剂

  1. 鼠胚胎成纤维细胞(MEF)
  2. 无酚红HBSS(Gibco ,目录号:14175-079)
  3. 4-(2-羟基乙基)-1-哌嗪乙磺酸(HEPES)(目录号:15630-080),</sup>
  4. 四甲基罗丹明甲酯(TMRM)(Life Technologies,目录号:T668)
  5. 环孢菌素H(Enzo Life Sciences,目录号:ALX-380-286-M001)
  6. Hoechst 33342
  7. 羰基氰化物 - 对三氟甲氧基苯基腙FCCP(Sigma-Aldrich,目录号:C2920-10MG)
  8. 胰蛋白酶(451) - 胰蛋白酶,0.05%(1X)与0.53mM EDTA 4Na,液体,20×100ml
    (Gibco ,目录号:25300096)

设备

  1. IN Cell Analyzer 1000(General Electric Company)
  2. 37℃5%CO 2细胞培养箱
  3. 12孔板(Corning,Costar ,目录号:CLS 3513)
  4. FACScan(BD)
  5. 离心机旋转速度:15,682,186×g(G力)(13,000rpm,半径8.3cm)

软件

  1. IN Cell Investigator分析软件(通用电气公司)
  2. BD CellQuest软件
  3. FlowJo软件

程序

  1. 用于定量实时分析
    1. 将细胞以50%细胞铺积铺板。
    2. 将细胞在37℃下在具有10mM HEPES,20nM TMRM,2μM环孢菌素-H,多药物抗性泵活性抑制剂但不具有渗透性转变的酚红色HBSS中孵育30分钟 孔(多药物抗性泵活性可影响用TMRM的线粒体上样)和2μg/ml Hoechst 33342,可用于活细胞的核染料。
    3. 在用于TMRM(535nm激发滤光片; 600nm发射)和Hoechst 33342(360nm激发滤光片; 460nm发射波长)的机动化方式中注射4μMFCCP(3个时间点)(3个时间点) )在不同的领域(每个孔获得4个视野,每个实验条件6个孔),每3分钟用IN CELL分析仪1000.
    4. 使用IN Cell Investigator分析软件自动分析图像以测量TMRM强度。在第一和最后一个时间点对每个场随机选择的10个点的平均值测量荧光强度,对于每个场去除对应于没有细胞的区域的背景。
    5. 在最后一个时间点(应用FCCP之后)测量的荧光强度通过在实验的第一时间点测量的荧光强度归一化。

  2. 用于FACS分析
    1. 将细胞铺在50%汇合
    2. 细胞被胰蛋白酶化(至少2百万个细胞)
    3. 将细胞在G力13,62下离心5分钟
    4. 将细胞在PBS中洗涤,重悬于含有10mM HEPES的无酚红的HBSS中并计数
    5. 在2μM多药抗性泵抑制剂环孢菌素-H存在下,将细胞(每管300,000个细胞,每个条件重复三次)重悬于含有10mM HEPES和20nM TMRM的无酚红的HBSS中,并温育30分钟 在37℃。 平行地,细胞在相同条件下用解偶联剂(4μMFCCP)温育5分钟以测量特异性线粒体染色。
    6. TMRM的FACS分析在具有CellQuest软件的BD FACScan上进行,并使用FlowJo软件分析。 通过门控活细胞计算TMRM的荧光强度
    7. FCCP的荧光强度通过没有FCCP的强度归一化,以排除强度差异仅仅或主要由细胞大小引起的可能性。

致谢

我们感谢文章的所有共同作者:Chiaravalli,M.,Mannella,V.,Ulisse,V.,Quilici,G.,Pema,M.,Song,X.W.,Xu,H。 Mari,S.,Qian,F.,Pei,Y。和Musco,G.,实验室的其他成员Boletta,Casari,G。和Cassina,L.和San Raffaele显微镜设备(Alembic)。

参考文献

  1. Rowe,I.,Chiaravalli,M.,Mannella,V.,Ulisse,V.,Quilici,G.,Pema,M.,Song,XW,Xu,H.,Mari,S.,Qian,F.,Pei ,Y.,Musco,G。和Boletta,A。(2013)。 多囊肾病中的葡萄糖代谢不足确定了一种新的治疗策略。 Med 19(4):488-493。
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Copyright: © 2017 The Authors; exclusive licensee Bio-protocol LLC.
引用:Rowe, I. and Boletta, A. (2013). Mitochondrial Transmembrane Potential (ψm) Assay Using TMRM. Bio-protocol 3(23): e987. DOI: 10.21769/BioProtoc.987.
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