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Isolating Brain Mitochondria by Differential Centrifugation
差速离心法分离脑线粒体   

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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 brain 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), Brain(脑), Respiration(呼吸), Bioenergetics(生物能学), Mice(老鼠)

Materials and Reagents

  1. Centrifuge tubes
  2. Mice
  3. Sucrose (Sigma-Aldrich, catalog number: 84100 )
  4. Bovine serum albumin (BSA) (Sigma-Aldrich, catalog number: A6003 )
  5. Ethylene glycol-bis(2-aminoethylether)-N, N, N’, N’-tetraacetic acid (EGTA) (Sigma-Aldrich, catalog number: E4378 )
  6. HEPES (Thermo Fisher Scientific, GibcoTM, catalog number: 15630-080 )
  7. Protease inhibitors (100x) (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: 78429 )
  8. Digitonin (Sigma-Aldrich, catalog number: D141 )
  9. D-Mannitol (Sigma-Aldrich, catalog number: M4125 )
  10. Magnesium chloride hexahydrate (MgCl2) (Sigma-Aldrich, catalog number: M9272 )
  11. Potassium hydroxide (KOH) (Sigma-Aldrich, catalog number: 221473 )
  12. Sodium hydroxide (NaOH) (Sigma-Aldrich, catalog number: S5881 )
  13. Extraction buffer (approximately 50 ml per brain) (see Recipes)

Equipment

  1. Dounce homogenizer and pestles (A and B)
  2. 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. Digitonin is added to disrupt the plasma membrane of synaptosomes and release synaptosomal mitochondria.
  4. Animal protocols must meet local ethics standards.

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

  2. Procedure
    1. Sacrifice the mouse by cervical dislocation, immediately remove the complete brain and place it in the ice-cold beaker with extraction buffer.
    2. Rinse the brain by adding and removing cold fresh buffer until most of the blood is removed (5-6 washes) (Figure 3).
    3. Mince the brain in the beaker in ice extensively using small scissors (Figure 4).
    4. Transfer the minced brain into a Dounce homogenizer (Figure 5) 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.
    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 in ice-cold extraction buffer with digitonin to a final concentration of 0.02% (Figure 7).
    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 0.1 ml) of extraction buffer or the specific experimental buffer (Figure 8).
      Notes:
      1. After isolation, protein concentration is determined by standard methods. Typically, around 2-3 milligrams of mitochondrial protein are obtained from one brain.
      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 brain


    Figure 4. Minced brain


    Figure 5. Homogenized brain


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


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


    Figure 8. Re-suspended mitochondria

Recipes

  1. Extraction buffer (freshly prepared)
    125 mM sucrose
    250 mM mannitol
    10 mM HEPES
    10 mM EGTA
    0.01% BSA
    1x protease inhibitors
    pH 7.2 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. 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.
  2. Tahara, E. B., Navarete, F. D. and Kowaltowski, A. J. (2009). Tissue-, substrate-, and site-specific characteristics of mitochondrial reactive oxygen species generation. Free Radic Biol Med 46(9): 1283-1297.

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。 该方案说明从小鼠脑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, 脑, 呼吸, 生物能学, 老鼠

材料和试剂

  1. 离心管
  2. 小鼠
  3. 蔗糖(Sigma-Aldrich,目录号:84100)
  4. 牛血清白蛋白(BSA)(Sigma-Aldrich,目录号:A6003)
  5. 乙二醇 - 双(2-氨基乙醚)-N,N,N',N'-四乙酸(EGTA)(Sigma-Aldrich,目录号:E4378)
  6. HEPES(Thermo Fisher Scientific,Gibco TM ,目录号:15630-080)
  7. 蛋白酶抑制剂(100x)(Thermo Fisher Scientific,Thermo Scientific TM ,目录号:78429)
  8. Digitonin(Sigma-Aldrich,目录号:D141)
  9. D-甘露醇(Sigma-Aldrich,目录号:M4125)
  10. 氯化镁六水合物(MgCl 2)(Sigma-Aldrich,目录号:M9272)
  11. 氢氧化钾(KOH)(Sigma-Aldrich,目录号:221473)
  12. 氢氧化钠(NaOH)(Sigma-Aldrich,目录号:S5881)
  13. 提取缓冲液(每只大脑约50ml)(见Recipes)

设备

  1. Dounce匀浆器和杵(A和B)
  2. 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. 添加了降钙素以破坏突触体的质膜并释放突触体线粒体。
  4. 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:此步骤是可选的,但会导致更重复的结果。
    2. 准备冰块容器并放置:
      1. Dounce匀浆器和杵(图1)
      2. 每个样品三个离心管(20-50 ml)
      3. 每个样品含有冷提取缓冲液的一个烧杯
    3. 将离心机和转子冷却至4℃
    4. 动物解剖区。
      1. 小剪刀(图1)
      2. 镊子(图1)

  2. 程序
    1. 牺牲小鼠颈椎脱位,立即取出 完整的大脑,并将其放在冰冷的烧杯中用提取 缓冲区
    2. 冲洗大脑通过添加和删除冷新鲜缓冲液,直到大多数血液被去除(5-6次洗涤)(图3)。
    3. 使用小剪刀将大脑放入冰中的烧杯中(图4)。
    4. 将切碎的脑转移到Dounce匀浆器中(图5),并加入约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
    6. 将匀浆转移到 离心管(图5),并用新鲜冷至30-40ml 提取缓冲液。 遵循差速离心步骤(图 2)。
    7. 在700×g和4℃离心10分钟。 将上清液倒入 新的冰冷管,并丢弃含有核和完整的丸 细胞(图6)。
    8. 重复该操作,在4℃下以700×g离心10分钟,然后将上清液倒入 新冰冷管。
    9. 在4℃下以10,000×g离心15分钟。 弃去上清液,并在冰冷的提取中重悬浮沉淀   缓冲液中至最终浓度为0.02%(图7)。
    10. 在4℃下以10,000×g离心15分钟,弃去 上清液,并在最小可能的情况下重悬最终的沉淀 体积(约0.1ml)提取缓冲液或具体实验   缓冲区(图8)。
      注意:
      1. 分离后,蛋白质 浓度通过标准方法测定。 通常,约2-3 从一个脑获得毫克的线粒体蛋白。
      2. 可以确定分离的线粒体的质量 呼吸控制比(RCR)使用氧电极和测量其在存在和不存在ADP的情况下的氧消耗速率。   RCR应当范围为4-6,丙酮酸加苹果酸和1.5-3与琥珀酸   加鱼藤酮。

    图2.通过差速离心的线粒体分离。 整个方案必须在4°C下进行。 避免过多的移液,通过反转转移上清液

    图3.提取的大脑


    图4.小脑


    图5.均匀化大脑


    图6.颗粒1(P1,原子核和完整细胞)


    图7.丸粒2(P2,富含线粒体的部分)


    图8.重悬线粒体

食谱

  1. 提取缓冲液(新鲜制备)
    125 mM蔗糖 250mM甘露糖 10 mM HEPES
    10mM EGTA
    0.01%BSA 1x蛋白酶抑制剂
    pH 7.2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,因为它防止钙从线粒体通过Na + /Ca sup> 2 + exchange。 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. 1. Rueda,CB,Traba,J.,Amigo,I.,Llorente-Folch,I.,Gonzalez-Sanchez,P.,Pardo,B.,Esteban,JA,del Arco,A.and Satrustegui,J。 2015)。 线粒体ATP-Mg/Pi载体SCaMC-3/Slc25a23抵消线粒体中PARP-1依赖性下降 ATP由神经元中的兴奋性毒性引起。 J Neurosci 35(8):3566-3581。 2.Tahara,E.B.,Navarete,F.D.and Kowaltowski,A.J。(2009)。 线粒体活性氧产生的组织特异性,底物特异性和位点特异性特征。 Free Radic Biol Med 46(9):1283-1297。
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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 Brain Mitochondria by Differential Centrifugation. Bio-protocol 6(10): e1810. DOI: 10.21769/BioProtoc.1810.
  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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Misha Koksharov
Northwestern University
Thank you for the detailed protocol and pictures. The homogenizer you show on the Figure 1 is actually not the classic Dounce version (which has a ball at the end) but a Tenbroeck homogenizer. Though, it's interesting how the performance of Dounce and Tenbroeck homogenizers compares.
10/26/2017 7:10:48 PM Reply