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Mouse Liver Mitochondria Isolation, Size Fractionation, and Real-time MOMP Measurement
小鼠肝脏线粒体分离、大小分级和实时外膜通透性(MOMP)测量   

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本实验方案简略版
Molecular Cell
Jan 2015

Abstract

The mitochondrial pathway of apoptosis involves a complex interplay between dozens of proteins and lipids, and is also dependent on the shape and size of mitochondria. The use of cellular models in past studies has not been ideal for investigating how the complex multi-factor interplay regulates the molecular mechanisms of mitochondrial outer membrane permeabilization (MOMP). Isolated systems have proven to be a paradigm to deconstruct MOMP into individual steps and to study the behavior of each subset of MOMP regulators. In particular, isolated mitochondria are key to in vitro studies of the BCL-2 family proteins, a complex family of pro-survival and pro-apoptotic proteins that directly control the mitochondrial pathway of apoptosis (Renault et al., 2013).

In this protocol, we describe three complementary procedures for investigating in real-time the effects of MOMP regulators using isolated mitochondria. The first procedure is “Liver mitochondria isolation” in which the liver is dissected from mice to obtain mitochondria. “Mitochondria labeling with JC-1 and size fractionation” is the second procedure that describes a method to label, fractionate by size and standardize subpopulations of mitochondria. Finally, the “Real-time MOMP measurements” protocol allows to follow MOMP in real-time on isolated mitochondria. The aforementioned procedures were used to determine in vitro the role of mitochondrial membrane shape at the level of isolated cells and isolated mitochondria (Renault et al., 2015).

Keywords: Apoptosis (细胞凋亡), Mitochondria (线粒体), BCL-2 Family (Bcl-2家族), MOMP (MOMP), Mitochondrial Fractionation (线粒体的分离)

Materials and Reagents

  1. 50 ml conical centrifuge tube (Santa Cruz Biotechnology, catalog number: sc-200251 )
  2. Petri dish, 100 x 15 mm (Fisher Scientific, catalog number: FB0875712 )
  3. 15 ml conical centrifuge tube (Santa Cruz Biotechnology, catalog number: sc-200250 )
  4. 1.5 ml Micro centrifuge tube (USA Scientific, catalog number: 1615-5510 )
  5. Gravity chromatography column (Thermo Fisher Scientific, PierceTM, catalog number: 29920 )
  6. Pasteur pipet (Santa Cruz Biotechnology, catalog number: sc-204537 )
  7. 96-well plate, flat bottom, black polystyrene (Corning, CostarTM, catalog number: 3915 )
  8. C57BL/6 mice (Charles River Laboratories, catalog number: 027 )
  9. 1x phosphate buffered saline (PBS), pH 7.4 (137 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, 2 mM KH2PO4) (Fisher Scientific, catalog number: BP24384 )
  10. Trehalose (Sigma-Aldrich, catalog number: 1673715 )
  11. Sucrose (Sigma-Aldrich, catalog number: S0389 )
  12. [4-(2-hydroxyethyl)piperazine-1-ethanesulfonic acid]-KOH (HEPES) (Sigma-Aldrich, catalog number: H0527 )
  13. KCl (Sigma-Aldrich, catalog number: P9541 )
  14. Ethylenediaminetetraacetic acid (EDTA) (Sigma-Aldrich, catalog number: E9884 )
  15. Ethyleneglycoltetraacetic acid (EGTA) (Sigma-Aldrich, catalog number: E3889 )
  16. Bovine serum albumin-fraction V (Sigma-Aldrich, catalog number: A9418 )
  17. Protease inhibitor cocktail (Thermo Fisher Scientific, HALTTM, catalog number: 78430 )
  18. Sepharose CL-2B resin (Sigma-Aldrich, catalog number: CL2B300 )
  19. 5,5’,6,6’-tetrachloro-1,1’,3,3’-tetraethylbenzimidazolylcarbocyanine iodide (JC-1) (Thermo Fisher Scientific, catalog number: T3168 )
  20. Triton X-100 (Fisher Scientific, catalog number: BP151-500 )
  21. Carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone (FCCP) (Sigma-Aldrich, catalog number: C2920 )
  22. Recombinant BAX [purified using the IMPACT (Intein Mediated Purification with an Affinity Chitin-binding Tag) system (New England Biolabs, catalog number: E6901S )]
  23. β-octylglucoside (OG) (Sigma-Aldrich, catalog number: O8001 )
  24. Dimethylsulfoxide (DMSO) (Fisher Scientific, catalog number: BP231-100 )
  25. Magnesium Chloride Hexahydrate (Fisher Scientific, catalog number: M33-500 )
  26. Trehalose isolation buffer (TIB) (see Recipes)
  27. JC-1 loading buffer (see Recipes)
  28. Liposome buffer (see Recipes)

Equipment

  1. Dissection tools (scissors, scalpel, forceps)
  2. Razor blade (Daigger Scientific, catalog number: EF7281A )
  3. 15 ml Potter-Elvehjem dounce homogenizer (Omni International, catalog number: 07-358044 )
  4. Swing bucket centrifuge (Thermo Fisher Scientific, Thermo ScientificTM SorvallTM, model: Legend XTR )
  5. Spectrophotometer (Fisher Scientific, GE Healthcare UltrospecTM, model: Ultrospec 7000 )
  6. Water bath (Fisher Scientific, IsoTempTM, model: 215 )
  7. Tabletop centrifuge (Thermo Fisher Scientific, SorvallTM LegendTM, model: Micro 21 )
  8. Fluorescence plate reader (Biotek, model: Synergy H1 )

Procedure

  1. Liver mitochondria isolation
    Animal handling, euthanasia, and dissection must be done following the Institutional Animal Care and Use Committee guidelines. We have successfully utilized carbon dioxide asphyxiation before cervical dislocation to euthanize the mice. However, we advise the reader to consult and follow the Animal Care and Use guidelines of their own institute for the appropriate procedure. Following steps involved in animal handling and dissection are executed wearing gloves.
    1. Visualize the liver, the bile duct, and gallbladder. The bile duct and gallbladder must be removed or kept separate from the liver to avoid contamination of the mitochondria with bile (Figure 1a-c).
    2. Excise the four liver lobes and transfer immediately to a 50 ml conical tube containing ice-cold PBS. In case of blood contamination, soak the liver in ice-cold PBS until no more blood is washed out (Figure 1d-e).
      Note: From this point, all the steps must be performed using reagents and materials at 4 °C to minimize the activation of proteases and phospholipases.


      Figure 1. Mouse dissection

    3. Transfer the liver with forceps to a clean Petri dish placed on ice and drain off the excess PBS.
    4. Mince the liver using a clean razor blade until forming a homogeneous paste (Figure 2).
    5. Transfer half of the liver paste using the razor blade as a spoon into a chilled Potter-Elvehjem dounce homogenizer containing 10 ml TIB.
    6. Insert the pestle into the homogenizer and gently push it downwards. Early resistance is common during the first strokes, and no excessive force should be applied to the liver paste. Instead, move the pestle upwards to resuspend the liver paste and slowly repeat those steps until all the material is able to go past the pestle without strong resistance. The homogenization step can be done on the bench as long the whole procedure is done quickly enough to maintain the temperature close to 4 °C.
    7. Homogenize the paste five times, pour into a 15 ml conical tube and store on ice until all the liver paste is processed (Figure 2).
    8. Repeat steps 6-8 with the second half of the liver paste.
    9. The following differential centrifugation steps are required to obtain the mitochondrial fraction. All the steps should be performed in a swinging bucket centrifuge at 4 °C. Resuspension of pellets is done gently by pipetting using a P1000 pipette (Figure 2).
      1. 600 x g for 10 min. Transfer the supernatant (S/N) to a clean, pre-chilled 15 ml conical tube.
      2. 3,500 x g for 10 min. Discard the S/N. Resuspend the pellet in 10 ml TIB.
      3. 1,500 x g for 5 min. Transfer the S/N to a clean, pre-chilled 15 ml conical tube.
      4. 5,500 x g for 10 min. Discard the S/N. Resuspend the pellet in 10 ml TIB.
      5. Repeat steps A9b to A9d, but resuspend the final pellet in 500 μl TIB.
    10. To quantify the amount of isolated material in the resuspended pellet (step A9e), add 5 μl of the resuspended pellet to 995 μl of TIB (1:200 dilution) and measure the OD520 with a spectrophotometer. Dilute the mitochondria with TIB to standardize the sample to an OD520 value of 0.25 (~20 μg/μl of protein).
    11. At this point the isolated mitochondria can be aliquoted (50 µl), frozen in dry ice-ethanol to prevent formation of water crystals, and stored at -80 °C.
      Note: Freshly isolated mitochondria from the previous step should be kept on ice and used within 2 h to ensure integrity of the outer membrane. If frozen mitochondria are used, thaw the sample in a 30 °C water bath and continue using TIB in the subsequent procedures.


      Figure 2. Liver mitochondria isolation

  2. Mitochondria labeling with JC-1 and size fractionation
    Labeling mitochondria with JC-1, a potential-dependent mitochondrial dye that changes color as membrane potentials increase during MOMP, allows to standardize the mitochondria fractions and to record MOMP in real-time (as example, see Renault et al., 2015, Figure 4F). The labeling and fractionation step offers the opportunity to select the mitochondria by size: big mitochondria will be eluted prior to smaller ones. This fractionation step is optional and requires to calibrate the column using liposomes of a known size as a standard. Another option is to determine the average mitochondrial size in each fraction a posteriori, using for example dynamic light scattering. If fractionation by size is not desired, all the mitochondria-containing fractions can be combined in step B8. 
    1. Dilute 50 µl of mitochondria in 250 µl TIB buffer.
    2. Add JC-1 (200 µM) to the diluted mitochondria to a final concentration of 15 µM and incubate for 10 min at 30 °C (Figure 3).


      Figure 3. Mitochondrial labeling and size fractionation

    3. To remove unbound JC-1, centrifuge the mitochondria at 5,550 x g for 10 min at room temperature and resuspend the pellet in 25 µl TIB.
    4. Pre-equilibrate a 2 ml CL-2B gravity-column with 2 column volumes of TIB (4 ml) using gravity to let the TIB to flow through the CL-2B resin, load the resuspended JC-1 mitochondria onto the column, and allow sample to flow through.
      Note: Make sure the resin does not run dry before loading the JC-1 labeled mitochondria.
    5. Slowly apply 4 column volumes of TIB (4 x 2 ml) to the column using a clean Pasteur pipet and collect 20 x 100 µl (~2-3 drops) fractions in micro-centrifuge tubes.
    6. To determine which fractions contain labeled mitochondria, pre-fill a 96-well plate with 95 µl of 0.1% Triton X-100 in water (0.1% Triton X-100 permeabilizes mitochondria) and add 5 µl sample of each fraction to a separate well.
    7. Measure the fluorescence using a spectrophotometer (Ex: 561 nm/Em: 620 nm) to identify the mitochondria-containing fractions (typical values are 10,000-50,000 Relative Fluorescence Units for big mitochondria, and > 500 RFU for small size mitochondria).
    8. Combine the desired fractions containing JC-1 labeled mitochondria and standardize the samples using relative JC-1 fluorescence intensity (determine the fluorescence of each combined sample as described in step B6-7 and dilute the most concentrated samples with TIB buffer to reach the concentration of the lowest sample).
      Note: In our experience 0.05-1 µm liposomes were subjected onto the CL-2B gravity-column and used as reference for selecting appropriate fractions. Briefly, fluorescent liposomes of several sizes (0.05, 0.2, and 1 µm) were prepared according to Asciolla et al., 2012. The liposomes were loaded onto the CL-2B gravity column and 100 µl fractions were collected, as described in steps B4-6 of this protocol. Identification of liposome-containing fractions for each liposome size allows to determine in which fractions vesicles of a given size should be expected.

  3. Real-time MOMP measurements
    Real-time MOMP quantification is determined by measuring the mitochondrial membrane potential (ΔΨM) using the fluorescent JC-1 dye. The JC-1 dye exhibits potential-dependent accumulation in mitochondria, indicated by a fluorescence emission shift from green (~529 nm) to red (~590 nm). MOMP and the subsequent loss of ΔΨM are indicated by a decrease in the red/green fluorescence intensity ratio. There is a plethora of options to investigate a subset of inducers and regulators of MOMP in real-time. It is important to have internal and experimental controls. An example is given in Table 1. For the positive internal control, we use FCCP that uncouples the electron transport from oxidative phosphorylation in mitochondria and completely depolarizes the mitochondrial membrane.
    To generate a positive experimental control, we use detergent-activated recombinant BAX (Hsu and Youle, 1997). The detergent, β-octylglucoside (OG), artificially triggers BAX activation, and therefore BAXOG (20-50 nM) can be used as a reliable positive control (for 100 µl of 2.3 µM BAXOG: 5 µg BAX + 0.7% OG in liposome buffer, incubate for 60 min at 4 °C, aliquot and store at -80 °C).
    Real-time measurements
    1. Prepare a 2x proteins/inducers solution in TIB and dispense 50 µl in each well in a 96-well plate. The plate should contain the following internal and experimental controls (Figure 4; Table 1)
      1. Negative internal control: TIB only
      2. Positive internal control: FCCP (10 µM final concentration)
      3. Negative experimental control: purified recombinant BAX (20-50 nM final concentration)
      4. Positive experimental control: β-octylglucoside-activated BAX (BAXOG, 20-50 nM final concentration)


      Figure 4. Real-Time MOMP

    2. Prepare a 2x suspension of JC-1 loaded mitochondria (~2.5 RFU/μl) and add 50 μl to each well to reach a final concentration of 1.25 RFU/μl.
      Note: ~50-100 RFUs per reaction is ideal.
    3. Measure fluorescence every 5 min in a plate reader for 60 min (Ex: 561 nm/Em: 620 nm) at 37 °C.
    4. Calculate the percentage of mitochondria that have undergone MOMP using the following formula:
      % MOMP = (RFUbuffer − RFUsample) / (RFUbuffer − RFUFCCP)
      Where RFUbuffer and RFUFCCP represent the fluorescence of the negative and positive internal controls, respectively, and RFUsample the fluorescence of the sample (see Figure 4G-H as example in Renault et al., 2015).

      Table 1. An example to measure MOMP in real-time with internal and experimental controls

Recipes

  1. Trehalose isolation buffer (TIB)
    200 mM trehalose
    68 mM sucrose
    10 mM HEPES-KOH, pH 7.4
    10 mM KCl
    1 mM EDTA
    1 mM EGTA
    0.1% BSA
    Protease inhibitors cocktail (freshly added according to manufacturer's instructions)
    Note: TIB should be prepared using BSA-Fraction V to eliminate fatty acid and lipid contaminants that promote non-specific BAK/BAX activation.
  2. JC-1 loading buffer
    200 μM stock solution is prepared in dimethylsulfoxide (DMSO) and diluted accordingly in TIB.
  3. Liposome buffer
    0.2 mM EDTA
    10 mM HEPES-KOH, pH 7.4
    200 mM KCl
    5 mM MgCl2

Acknowledgments

We would like to thank everyone in the Chipuk Laboratory for their assistance and support. This work was supported by the following: NIH grants CA157740 and CA206005 (to J.E. Chipuk.); a pilot project from NIH P20AA017067 (to J.E. Chipuk), the JJR Foundation (to J.E. Chipuk), the William A. Spivak Fund (to J.E. Chipuk), the Fridolin Charitable Trust (to J.E. Chipuk), and an American Cancer Society Research Scholar Award (to J.E. Chipuk). This work was also supported in part by two research grants (5-FY11-74 and 1-FY13-416) from the March of Dimes Foundation (to J.E. Chipuk), the Leukemia and Lymphoma Scholar Award (to J.E. Chipuk), and the Developmental Research Pilot Project Program within the Department of Oncological Sciences at Mount Sinai (to J.E. Chipuk).

References

  1. Asciolla, J. J., Renault, T. T. and Chipuk, J. E. (2012). Examining BCL-2 family function with large unilamellar vesicles. J Vis Exp (68).
  2. Hsu, Y. T. and Youle, R. J. (1997). Nonionic detergents induce dimerization among members of the Bcl-2 family. J Biol Chem 272(21): 13829-13834.
  3. Renault, T. T., Floros, K. V. and Chipuk, J. E. (2013). BAK/BAX activation and cytochrome c release assays using isolated mitochondria. Methods 61(2): 146-155.
  4. Renault, T. T., Floros, K. V., Elkholi, R., Corrigan, K. A., Kushnareva, Y., Wieder, S. Y., Lindtner, C., Serasinghe, M. N., Asciolla, J. J., Buettner, C., Newmeyer, D. D. and Chipuk, J. E. (2015). Mitochondrial shape governs BAX-induced membrane permeabilization and apoptosis. Mol Cell 57(1): 69-82.

简介

凋亡的线粒体途径涉及数十种蛋白质和脂质之间的复杂相互作用,并且还依赖于线粒体的形状和大小。在过去的研究中使用细胞模型不是理想的调查如何复杂的多因素相互作用调节线粒体外膜透化(MOMP)的分子机制。分离系统已被证明是将MOMP解构成各个步骤并研究每个子集的MOMP调节剂的行为的范例。特别地,分离的线粒体是BCL-2家族蛋白的体外研究的关键,BCL-2家族蛋白是直接控制凋亡的线粒体途径的促存活和促凋亡蛋白的复合家族(Renault > et al 。,2013)。
  在这个协议,我们描述三个补充程序用于实时调查使用孤立的线粒体MOMP调节器的影响。第一种方法是"肝线粒体分离",其中从小鼠中分离肝脏以获得线粒体。 "用JC-1和大小分级分离的线粒体标记"是描述标记,按大小分级并标准化线粒体亚群的方法的第二个方法。最后,"实时MOMP测量"协议允许在孤立的线粒体上实时跟踪MOMP。上述程序用于在体外确定线粒体膜形状在分离的细胞和分离的线粒体水平上的作用(Renault等人,2015)。

关键字:细胞凋亡, 线粒体, Bcl-2家族, MOMP, 线粒体的分离

材料和试剂

  1. 50ml锥形离心管(Santa Cruz Biotechnology,目录号:sc-200251)
  2. 培养皿,100×15mm(Fisher Scientific,目录号:FB0875712)
  3. 15ml锥形离心管(Santa Cruz Biotechnology,目录号:sc-200250)
  4. 1.5ml微量离心管(USA Scientific,目录号:1615-5510)
  5. 重力色谱柱(Thermo Fisher Scientific,Pierce TM,目录号:29920)
  6. 巴斯德吸管(Santa Cruz Biotechnology,目录号:sc-204537)
  7. 96孔板,平底,黑色聚苯乙烯(Corning,Costar TM ,目录号:3915)
  8. C57BL/6小鼠(Charles River Laboratories,目录号:027)
  9. 1×磷酸盐缓冲盐水(PBS),pH 7.4(137mM NaCl,2.7mM KCl,10mM Na 2 HPO 4,2mM KH 2, > PO 4)(Fisher Scientific,目录号:BP24384)
  10. 海藻糖(Sigma-Aldrich,目录号:1673715)
  11. 蔗糖(Sigma-Aldrich,目录号:SO389)
  12. [4-(2-羟乙基)哌嗪-1-乙烷磺酸] -KOH(HEPES)(Sigma-Aldrich,目录号:H0527)
  13. KCl(Sigma-Aldrich,目录号:P9541)
  14. 乙二胺四乙酸(EDTA)(Sigma-Aldrich,目录号:E9884)
  15. 乙二醇四乙酸(EGTA)(Sigma-Aldrich,目录号:E3889)
  16. 牛血清白蛋白馏分V(Sigma-Aldrich,目录号:A9418)
  17. 蛋白酶抑制剂混合物(Thermo Fisher Scientific,HALT TM ,目录号:78430)
  18. Sepharose CL-2B树脂(Sigma-Aldrich,目录号:CL2B300)
  19. 5,5'6,6'-四氯-1,1',3,3'-四乙基苯并咪唑基羰花青碘化物(JC-1)(Thermo Fisher Scientific,目录号:T3168)
  20. Triton X-100(Fisher Scientific,目录号:BP151-500)
  21. 羰基氰4-(三氟甲氧基)苯腙(FCCP)(Sigma-Aldrich,目录号:C2920)
  22. 重组BAX [使用IMPACT(具有亲和壳多糖结合标签的Intein Mediated Purification)系统(New England Biolabs,目录号:E6901S)纯化]
  23. β-辛基葡糖苷(OG)(Sigma-Aldrich,目录号:O8001)
  24. 二甲基亚砜(DMSO)(Fisher Scientific,目录号:BP231-100)
  25. 六水合氯化镁(Fisher Scientific,目录号:M33-500)
  26. 海藻糖分离缓冲液(TIB)(参见配方)
  27. JC-1加载缓冲区(参见配方)
  28. 脂质体缓冲液(参见配方)

设备

  1. 解剖工具(剪刀,解剖刀,镊子)
  2. 剃刀刀片(Daigger Scientific,目录号:EF7281A)
  3. 15ml Potter-Elvehjem dounce匀浆器(Omni International,目录号:07-358044)
  4. 回转桶离心机(Thermo Fisher Scientific,Thermo Scientific TM Sorvall TM ,型号:Legend XTR)
  5. 分光光度计(Fisher Scientific,GE Healthcare Ultrospec TM,型号:Ultrospec 7000)
  6. 水浴(Fisher Scientific,IsoTemp TM,型号:215)
  7. 台式离心机(Thermo Fisher Scientific,Sorvall Legend TM ,型号:Micro 21)
  8. 荧光平板读数器(Biotek,型号:Synergy H1)

程序

  1. 肝线粒体隔离
    动物处理,安乐死和解剖必须根据机构动物护理和使用委员会指南进行。我们已经成功地利用二氧化碳窒息在颈椎脱臼之前使小鼠安乐死。但是,我们建议读者咨询并遵守其自己的研究所的动物护理和使用指南以进行适当的操作。以下涉及动物处理和解剖的步骤用手套进行
    1. 可视化肝脏,胆管和胆囊。胆管和胆囊必须被去除或保持与肝脏分开,以避免线粒体被胆汁污染(图1a-c)。
    2. 切除四个肝叶,立即转移到含有冰冷PBS的50ml锥形管中。在血液污染的情况下,将肝浸泡在冰冷的PBS中,直到没有更多的血液被洗出(图1d-e)。
      注意:从这一点来说,所有的步骤都必须使用试剂和材料在4℃下进行,以尽量减少蛋白酶和磷脂酶的活化。


      图1.鼠标解剖

    3. 用镊子转移肝脏到一个干净的培养皿放在冰上,排出多余的PBS
    4. 使用干净的剃刀刀片将肝脏切碎,直到形成均匀的糊状物(图2)
    5. 使用剃须刀刀片将一半肝膏转移到含有10ml TIB的冷却的Potter-Elvehjem dounce匀浆器中。
    6. 将杵插入均质器,轻轻向下推。早期阻力在第一次冲击期间是常见的,并且不应向肝膏施加过大的力。相反,将杵向上移动以重新悬浮肝脏膏,并缓慢地重复这些步骤,直到所有的材料能够通过杵没有强烈的阻力。均匀化步骤可以在台架上进行,只要整个程序足够快地进行以保持温度接近4℃即可。
    7. 将糊状物均匀化5次,倒入15ml锥形管中并储存在冰上,直到所有肝脏糊状物被处理(图2)。
    8. 重复步骤6-8与肝脏的后半部分粘贴。
    9. 需要以下差速离心步骤来获得线粒体级分。所有步骤应在4℃的摇桶式离心机中进行。通过使用P1000移液管吸移轻轻地重悬沉淀物(图2)
      1. 600 x g 10分钟。将上清液(S/N)转移到一个干净的,预冷的15毫升锥形管
      2. 3,500 x g 10分钟。丢弃S/N。将沉淀重悬在10ml TIB中
      3. 1,500 x g 5分钟。将S/N转移到一个干净的,预冷的15毫升锥形管
      4. 5,500 x g 10分钟。丢弃S/N。将沉淀重悬在10ml TIB中
      5. 重复步骤A9b到A9d,但将最终沉淀重悬在500μlTIB中
    10. 为了定量重悬沉淀物中分离的物质的量(步骤A9e),将5μl重悬浮的沉淀物加入到995μlTIB(1:200稀释)中,并用分光光度计测量OD520。用TIB稀释线粒体以将样品标准化至OD520值为0.25(?20μg/μl蛋白质)。
    11. 此时,可以将分离的线粒体等分(50μl),在干冰 - 乙醇中冷冻以防止形成水晶,并储存在-80℃。
      注意:来自前一步骤的新鲜分离的线粒体应该保持在冰上并在2小时内使用以确保外膜的完整性。如果使用冷冻线粒体,在30℃水浴中解冻样品,并在后续程序中继续使用TIB。


      图2.肝线粒体隔离

  2. 线粒体标记用JC-1和大小分级
    用JC-1标记线粒体,JC-1是在MOMP期间膜电位增加时改变颜色的电位依赖性线粒体染料,允许标准化线粒体级分并实时记录MOMP(例如,参见Renault等人, ,2015,图4F)。标记和分馏步骤提供了通过尺寸选择线粒体的机会:大线粒体将在较小的线粒体之前被洗脱。该分馏步骤是任选的,并且需要使用已知尺寸的脂质体作为标准校准柱。另一个选择是使用例如动态光散射来确定每个分数中的平均线粒体大小。如果不希望按大小分级,则可以在步骤B8中组合所有含线粒体的级分。
    1. 在250μlTIB缓冲液中稀释50μl线粒体
    2. 将JC-1(200μM)加入稀释的线粒体中至终浓度为15μM,并在30℃下孵育10分钟(图3)。


      图3.线粒体标记和大小分离

    3. 为了除去未结合的JC-1,在室温下将线粒体以5,550×g离心10分钟,并将沉淀重悬于25μlTIB中。
    4. 使用重力使2ml CL-2B重力柱与2个柱体积的TIB(4ml)预平衡以使TIB流过CL-2B树脂,将重悬的JC-1线粒体装载到柱上,并允许样品流过。
      注意:确保树脂在装入JC-1标记的线粒体前不会变干。
    5. 使用干净的巴斯德吸管缓慢地将4个柱体积的TIB(4×2ml)施加到柱上,并在微量离心管中收集20×100μl(?2-3滴)级分。
    6. 为了确定哪些级分含有标记的线粒体,用95μl的0.1%Triton X-100水溶液(0.1%Triton X-100透化线粒体)预填充96孔板,并将每个级分的5μl样品加入到单独的孔。
    7. 使用分光光度计(Ex:561nm/Em:620nm)测量荧光以鉴定含线粒体的级分(典型值为大线粒体的10,000-50,000相对荧光单位,对于小尺寸线粒体大于500RFU) br />
    8. 合并含有JC-1标记的线粒体的所需级分,并使用相对JC-1荧光强度标准化样品(确定如步骤B6-7中所述的每个组合样品的荧光,并用TIB缓冲液稀释最浓缩的样品以达到最低样本)。
      注意:根据我们的经验,将0.05-1μm脂质体置于CL-2B重力柱上,并用作选择合适级分的参考。简言之,根据Asciolla等人,2012制备几种尺寸(0.05,0.2和1μm)的荧光脂质体。将脂质体加载到CL-2B重力柱上,并收集100μl级分,如步骤B4 -6。鉴定每个脂质体大小的含有脂质体的级分允许确定应该预期在哪个级分中具有给定大小的泡囊。

  3. 实时MOMP测量
    实时MOMP定量通过使用荧光JC-1染料测量线粒体膜电位(ΔΨM)来确定。 JC-1染料显示出线粒体中的电位依赖性积聚,由从绿色(?529nm)到红色(?590nm)的荧光发射移动指示。 MOMP和随后的ΔΨM的损失由红/绿荧光强度比的降低指示。有大量的选项,以实时调查MOMP的诱导物和调节器的子集。有内部和实验控制是重要的。在表1中给出了一个实例。对于阳性内部对照,我们使用FCCP,其将线粒体中的氧化磷酸化的电子传递解耦并完全去极化线粒体膜。
    为了产生阳性实验对照,我们使用洗涤剂活化的重组BAX(Hsu和Youle,1997)。洗涤剂β-辛基葡糖苷(OG)人为引发BAX活化,因此BAX sup OG(20-50nM)可用作可靠的阳性对照(对于100μl的2.3μMBAX OG:在脂质体缓冲液中的5μgBAX + 0.7%OG,在4℃孵育60分钟,等分并保存在-80℃)。
    实时测量
    1. 在TIB中制备2x蛋白/诱导剂溶液,并在96孔板中的每个孔中分配50μl。板应包含以下内部和实验控制(图4;表1)
      1. 阴性内部控制:仅TIB
      2. 阳性内部对照:FCCP(10μM终浓度)
      3. 阴性实验对照:纯化的重组BAX(最终浓度为20-50nM)
      4. 阳性实验对照:β-辛基葡糖苷 - 活化的BAX(BAX OG ,最终浓度为20-50nM)


      图4.实时MOMP

    2. 准备一个2x悬浮的JC-1负载线粒体(?2.5 RFU /μl),并添加50μl到每个孔,以达到1.25 RFU /μl的最终浓度。
      注意:每个反应?50-100个RFU是理想的。
    3. 在平板读数器中在37℃下每5分钟测量荧光60分钟(Ex:561nm/Em:620nm)。
    4. 使用以下公式计算已经历MOMP的线粒体的百分比:
      %MOMP =(RFU buffer - RFU sample )/(RFU buffer - RFU sub FCCP )
      其中RFU buffer和RFU FCCP 分别代表阴性和阳性内部对照的荧光,RFU 样品图4G-H作为例子在雷诺等人。,2015年)。

      表1.使用内部和实验控件实时测量MOMP的示例

食谱

  1. 海藻糖分离缓冲液(TIB)
    200mM海藻糖 68mM蔗糖 10mM HEPES-KOH,pH7.4 10 mM KCl
    1mM EDTA
    1 mM EGTA
    0.1%BSA
    蛋白酶抑制剂混合物(根据制造商的说明新鲜加入)
    注意:应使用BSA-Fraction V制备TIB以消除促进非特异性BAK/BAX激活的脂肪酸和脂质污染物。
  2. JC-1加载缓冲区
    在二甲基亚砜(DMSO)中制备200μM储备溶液,并相应地在TIB中稀释
  3. 脂质体缓冲液
    0.2 mM EDTA
    10mM HEPES-KOH,pH7.4 200 mM KCl
    5mM MgCl 2/

致谢

我们要感谢Chipuk实验室的所有人的帮助和支持。这项工作由以下支持:NIH授予CA157740和CA206005(到J.E.Chipuk。一个来自NIH P20AA017067(JE Chipuk),JJR基金会(JE Chipuk),William A.Spivak基金(JE Chipuk),Fridolin慈善基金会(JE Chipuk)和美国癌症学会研究学者奖(JE Chipuk)。这项工作还得到了3月的Dime基金会(JE Chipuk),白血病和淋巴瘤学者奖(JE Chipuk)的两项研究资助(5 - FY11-74和1 - FY13-416),以及西奈山肿瘤科学系的发展研究试点项目(JE Chipuk)。

参考文献

  1. Asciolla,JJ,Renault,TT和Chipuk,JE(2012)。  检查具有大单层囊泡的BCL-2家族功能。 (68)。
  2. Hsu,YT和Youle,RJ(1997)。  非离子洗涤剂在Bcl-2家族的成员之间诱导二聚化。 J Biol Chem 272(21):13829-13834。
  3. Renault,TT,Floros,KV and Chipuk,JE(2013)。  使用分离的线粒体的BAK/BAX激活和细胞色素c释放测定。 61(2):146-155。
  4. Renault,TT,Floros,KV,Elkholi,R.,Corrigan,KA,Kushnareva,Y。,Wieder,SY,Lindtner,C.,Serasinghe,MN,Asciolla,JJ,Buettner,C.,Newmeyer,DDand Chipuk, JE(2015)。  线粒体形状控制BAX诱导的膜透化和细胞凋亡。 Mol Cell 57(1):69-82
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Copyright: © 2016 The Authors; exclusive licensee Bio-protocol LLC.
引用:Renault, T. T., Luna-Vargas, M. P. and Chipuk, J. E. (2016). Mouse Liver Mitochondria Isolation, Size Fractionation, and Real-time MOMP Measurement. Bio-protocol 6(15): e1892. DOI: 10.21769/BioProtoc.1892.
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