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Isolating Liver Mitochondria by Differential Centrifugation
差速离心法分离肝脏线粒体   

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Abstract

In addition to methods aimed at the study of mitochondrial function in-situ, a full understanding of mitochondrial function requires their purification from cells or tissues under specific physiological or pathological conditions. This protocol illustrates a sequential procedure to obtain functional mitochondria with high yield from mice liver tissue. Mitochondria obtained with this method can be used to assess different mitochondrial parameters, including oxygen consumption, membrane potential and calcium retention capacity.

Keywords: Mitochondria(线粒体), Liver(肝), Respiration(呼吸), Bioenergetics(生物能学), Mice(老鼠)

Materials and Reagents

  1. Centrifuge tubes
  2. Mice
  3. Potassium salts
  4. Sodium salts
  5. Sucrose (Sigma-Aldrich, catalog number: 84100 )
  6. Bovine serum albumin (BSA) (Sigma-Aldrich, catalog number: A6003 )
  7. Disodium ethylenediaminetetraacetate dihydrate (EDTA) (Sigma-Aldrich, catalog number: ED2SS )
  8. Ethylene glycol-bis(2-aminoethylether)-N, N, N’, N’-tetraacetic acid (EGTA) (Sigma-Aldrich, catalog number: E4378 )
  9. Dithiothreitol (DTT) (Sigma-Aldrich, catalog number: 646563 )
  10. HEPES (Thermo Fisher Scientific, GibcoTM, catalog number: 15630-080 )
  11. Protease inhibitors (100x) (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: 78429 )
  12. D-Mannitol (Sigma-Aldrich, catalog number: M4125 )
  13. Magnesium chloride hexahydrate (MgCl2) (Sigma-Aldrich, catalog number: M9272 )
  14. Potassium hydroxide (KOH) (Sigma-Aldrich, catalog number: 221473 )
  15. Sodium hydroxide (NaOH) (Sigma-Aldrich, catalog number: S5881 )
  16. Extraction buffer (see Recipes)
    Note: Approximately 100 ml extraction buffer per liver.

Equipment

  1. Dounce homogenizer and pestles (A and B)
  2. Small scissors
  3. Tweezers


    Figure 1. Tools for mincing and homogenate the tissue. Dounce homogenizer and pestles (A and B), Small scissors and tweezers.

Procedure

Notes:

  1. To obtain a high yield of functional mitochondria the procedure must be done as fast as possible (preferably in less than 1.5 h) and the samples maintained on ice at all times.
  2. The procedure described here refers to mice but can be used in rats by adapting the volumes. Functional assays should be carried out within 3-4 h of isolation.
  3. Buffers can be made using potassium or sodium salts depending on the use of the mitochondria.
  4. If potassium can affect the subsequent mitochondrial experiments, KCl can be replaced by mannitol.
  5. Animal protocols must meet local ethics standards.

  1. Preparation
    1. Starve animals overnight (8-16 h during the dark cycle)
      Note: Starvation is optional but leads to more reproducible results, especially in the liver.
    2. Prepare a container with ice and place:
      1. Dounce homogenizer and pestles
      2. Three centrifuge tubes per sample (20-50 ml)
      3. One beaker containing extraction buffer per sample
    3. Cool centrifuge and rotor to 4 °C. Cool on ice Dounce and pestles (Figure 1).
    4. Animal dissection material (Figure 1)
      1. Small scissors
      2. Tweezers

  2. Procedure
    1. Sacrifice the mouse by cervical dislocation, immediately remove the liver and place it in the ice-cold beaker with extraction buffer.
    2. Rinse the liver by adding and removing cold fresh extraction buffer until most of the blood is removed (5-6 washes).
    3. Mince the liver in the beaker in ice extensively using small scissors until homogeneus (Figure 3)
    4. Transfer the minced liver into a Dounce homogenizer and add approximately 3 ml of cold extraction buffer.
    5. With the homogenizer placed in the ice container, gently grind the tissue ten times with the A pestle (looser) and another ten with the B pestle (tighter). Avoiding the formation of bubbles is critical to obtain high quality mitochondria (Figure 4).
    6. Transfer the homogenate into a centrifuge tube (Figure 5) and complete to 30-40 ml with fresh cold extraction buffer. Follow the differential centrifugation steps (Figure 2)
    7. Centrifuge 10 min at 700 x g and 4 °C. Pour supernatant to a new ice-cold tube and discard the pellet containing nuclei and intact cells (Figure 6).
    8. Repeat the operation centrifuging again at 700 x g for 10 min at 4 °C and subsequently pouring the supernatant to a new ice-cold tube.
    9.  Centrifuge at 10,000 x g for 15 min at 4 °C. Discard the supernatant and re-suspend the pellet (Figure 7) in ice-cold extraction buffer.
    10. Centrifuge at 10,000 x g for 15 min at 4 °C, discard the supernatant and re-suspend the final pellet in the minimal possible volume (around 0.3 ml) of extraction buffer or the specific experimental buffer (Figure 8).
      Notes:
      1. After isolation, protein concentration is determined by standard methods. Typically, around 30-40 mg of mitochondrial protein are obtained from one liver.
      2. The quality of the isolated mitochondria can be determined their respiratory control ratio (RCR) using an oxygen electrode and measuring their oxygen consumption rate in the presence and in the absence of ADP. RCR should range 4-6 with pyruvate plus malate and 1.5-3 with succinate plus rotenone.


    Figure 2. Mitochondrial isolation by differential centrifugation. The whole protocol must be carried at 4 °C. Avoid excessive pipetting, transfer supernatants by inversion.


    Figure 3. Extracted liver


    Figure 4. Minced liver


    Figure 5. Homogenized liver


    Figure 6. Pellet 1 (P1, nuclei and intact cells)


    Figure 7. Pellet 2 (P2, mitochondrial enriched fraction)


    Figure 8. Resuspended mitochondria

Recipes

  1. Extraction buffer (freshly prepared)
    250 mM sucrose
    250 mM mannitol
    25 mM HEPES
    10 mM KCl
    0.25 mM EDTA
    10 mM EGTA
    1.5 mM MgCl2
    1 mM DTT
    0.1% BSA
    1x protease inhibitors
    pH 7.4 with KOH or NaOH
    Note: The type of salt used can interfere with some functional assays. KOH is recommended for calcium handling experiments, as it prevents the efflux of calcium from the mitochondria through the Na+/Ca2+ exchanger. For membrane potential experiments using safranin O, NaOH is recommended to allow calibration with KOH and valinomycin.

Acknowledgments

We thank Dr. Jorgina Satrustegui, in whose laboratory our previous work was carried out, and Dr. Araceli del Arco, for constant help, guidance, support and critical comments.

References

  1. Amigo, I., Traba, J., Gonzalez-Barroso, M. M., Rueda, C. B., Fernandez, M., Rial, E., Sanchez, A., Satrustegui, J. and Del Arco, A. (2013). Glucagon regulation of oxidative phosphorylation requires an increase in matrix adenine nucleotide content through Ca2+ activation of the mitochondrial ATP-Mg/Pi carrier SCaMC-3. J Biol Chem 288(11): 7791-7802.
  2. Frezza, C., Cipolat, S. and Scorrano, L. (2007). Organelle isolation: functional mitochondria from mouse liver, muscle and cultured fibroblasts. Nat Protoc 2(2): 287-295.

简介

除了旨在研究线粒体功能的方法之外,充分了解线粒体功能需要在特定生理或病理条件下从细胞或组织中纯化。 该方案说明从小鼠肝组织获得高产量的功能性线粒体的顺序程序。 用该方法获得的线粒体可用于评估不同线粒体参数,包括氧消耗,膜电位和钙保留能力。

关键字:线粒体, 肝, 呼吸, 生物能学, 老鼠

材料和试剂

  1. 离心管
  2. 小鼠
  3. 钾盐
  4. 钠盐
  5. 蔗糖(Sigma-Aldrich,目录号:84100)
  6. 牛血清白蛋白(BSA)(Sigma-Aldrich,目录号:A6003)
  7. 乙二胺四乙酸二钠二水合物(EDTA)(Sigma-Aldrich,目录号:ED2SS)
  8. 乙二醇 - 双(2-氨基乙醚)-N,N,N',N'-四乙酸(EGTA)(Sigma-Aldrich,目录号:E4378)
  9. 二硫苏糖醇(DTT)(Sigma-Aldrich,目录号:646563)
  10. HEPES(Thermo Fisher Scientific,Gibco TM ,目录号:15630-080)
  11. 蛋白酶抑制剂(100x)(Thermo Fisher Scientific,Thermo Scientific TM ,目录号:78429)
  12. D-甘露醇(Sigma-Aldrich,目录号:M4125)
  13. 氯化镁六水合物(MgCl 2)(Sigma-Aldrich,目录号:M9272)
  14. 氢氧化钾(KOH)(Sigma-Aldrich,目录号:221473)
  15. 氢氧化钠(NaOH)(Sigma-Aldrich,目录号:S5881)
  16. 提取缓冲液(参见配方)
    注意:每个肝脏大约提取100ml提取缓冲液。

设备

  1. Dounce匀浆器和杵(A和B)
  2. 小剪刀
  3. 镊子


    图1.用于切碎和匀浆组织的工具。Dounce均质器和杵(A和B),小剪刀和镊子。

程序

注意:

  1. 为了获得高产量的功能性线粒体,程序必须   尽可能快地(优选在小于1.5小时内)和 样品始终保持在冰上。
  2. 程序 本文所述的术语"小鼠"是指小鼠,但可以通过适应大鼠使用 卷。 功能测定应在3-4小时内进行 隔离。
  3. 根据线粒体的使用,可以使用钾盐或钠盐制备缓冲液。
  4. 如果钾可以影响随后的线粒体实验,KCl可以被甘露醇替代。
  5. 动物协议必须符合当地道德标准。

  1. 准备
    1. 饥饿动物过夜(黑暗周期中8-16小时)
      注意:饥饿是可选的,但会导致更重复的结果,特别是在肝脏。
    2. 准备冰块容器并放置:
      1. Dounce匀浆器和杵
      2. 每个样品三个离心管(20-50 ml)
      3. 每个样品含有提取缓冲液的一个烧杯
    3. 将离心机和转子冷却至4℃。 在冰上冷却Dounce和杵(图1)
    4. 动物解剖材料(图1)
      1. 小剪刀
      2. 镊子

  2. 程序
    1. 牺牲小鼠颈椎脱位,立即取出 肝脏,并将其放在冰冷的烧杯中用提取缓冲液
    2. 通过添加和去除冷的新鲜提取缓冲液冲洗肝脏,直到大部分血液被去除(5-6次洗涤)。
    3. 使用小剪刀将烧杯中的肝脏广泛地剁碎,直到均匀(图3)
    4. 将切碎的肝脏转移到Dounce匀浆器中,加入约3ml冷提取缓冲液
    5. 用均化器放置在冰容器中,轻轻研磨 组织十次用A杵(松)和另一个十与B 杵(更紧)。 避免气泡的形成至关重要 获得高质量的线粒体(图4)
    6. 转移 匀浆至离心管中(图5),并完成至30-40ml用新鲜冷提取缓冲液。 跟随差分 离心步骤(图2)
    7. 在700×g离心10分钟和4   C。 将上清液倒入新的冰冷管中并弃去沉淀 含有核和完整细胞(图6)。
    8. 重复 在4℃下以700×g离心10分钟再次操作离心 随后将上清液倒入新的冰冷管中。
    9.  在4℃下以10,000×g离心15分钟。 弃去上清液   在冰冷的提取缓冲液中重悬浮沉淀(图7)。
    10. 在4℃下以10,000×g离心15分钟,弃去上清液并以最小可能体积(周围)重悬最终的沉淀 0.3 ml)的提取缓冲液或特定的实验缓冲液   8)。
      注意:
      1. 分离后,蛋白质浓度为 通过标准方法测定。 通常,约30-40mg 线粒体蛋白从一个肝脏获得。
      2. 质量 的孤立线粒体可以确定他们的呼吸控制   比率(RCR),并测量它们的氧 在存在和不存在ADP的情况下的消耗率。 RCR应该 范围4-6与丙酮酸加苹果酸和1.5-3与琥珀酸加 鱼藤酮。


    图2.通过差速离心进行的线粒体分离。整个方案必须在4℃下进行。避免过多的移液,通过倒置转移上清液

    图3.摘出的肝脏


    图4.碎肝


    图5.均质肝脏


    图6.颗粒1(P1,核和完整细胞)


    图7.粒2(P2,富含线粒体的部分)


    图8.重悬的线粒体

食谱

  1. 提取缓冲液(新鲜制备)
    250mM蔗糖 250mM甘露糖 25 mM HEPES
    10 mM KCl
    0.25mM EDTA 10mM EGTA
    1.5mM MgCl 2·h/v1 mM DTT
    0.1%BSA
    1x蛋白酶抑制剂
    pH 7.4用KOH或NaOH洗涤 注意:所用盐的类型可能会干扰一些功能测定。 KOH被推荐用于钙处理实验,因为其防止钙从线粒体通过Na +/Ca +/Ca 2+交换器流出。 对于使用safranin O的膜电位实验,建议NaOH允许用KOH和缬氨霉素校准。

致谢

我们感谢Jorgina Satrustegui博士,我们以前的工作在其实验室进行,Araceli del Arco博士,持续的帮助,指导,支持和批评性评论。

参考文献

  1. Amigo,I.,Traba,J.,Gonzalez-Barroso,M.M.,Rueda,C.B.,Fernandez,M.,Rial,E.,Sanchez,A.,Satrustegui,J.and Del Arco, 胰高血糖素调节氧化磷酸化需要通过Ca 2+ 增加基质腺嘌呤核苷酸含量,/sup>激活线粒体ATP-Mg/Pi载体SCaMC-3。 J Biol Chem 288(11):7791-7802。
  2. Frezza,C.,Cipolat,S.and Scorrano,L。(2007)。 细胞器分离:来自小鼠肝脏,肌肉和培养的成纤维细胞的功能性线粒体。 Nat Protoc 2(2):287-295
  • English
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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: © 2016 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. Amigo, I., Traba, J. and Rueda, C. B. (2016). Isolating Liver Mitochondria by Differential Centrifugation. Bio-protocol 6(10): e1809. DOI: 10.21769/BioProtoc.1809.
  2. Rueda, C. B., Traba, J., Amigo, I., Llorente-Folch, I., Gonzalez-Sanchez, P., Pardo, B., Esteban, J. A., del Arco, A. and Satrustegui, J. (2015). Mitochondrial ATP-Mg/Pi carrier SCaMC-3/Slc25a23 counteracts PARP-1-dependent fall in mitochondrial ATP caused by excitotoxic insults in neurons. J Neurosci 35(8): 3566-3581.
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