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Mitochondrial Isolation and Purification from Mouse Spinal Cord
小鼠脊髓的线粒体分离和纯化   

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Abstract

Mitochondria are eukaryotic organelles that play a crucial role in several cellular processes, including energy production, β-oxidation of fatty acids and regulation of calcium homeostasis. In the last 20 years there has been a hightened interest in the study of mitochondria following the discoveries that mitochondria are central to the process of programmed cell death and that mitochondrial dysfunctions are implicated in numerous diseases including a wide range of neurological disorders such as Parkinson’s disease, Alzheimer’s disease, Huntington’s disease and amyotrophic lateral sclerosis. In order to identify and study changes in mitochondrial function related to specific neurological conditions the mitochondria are often isolated from the compartment of the central nervous system most affected during disease. Here, we describe a protocol for the isolation of mitochondria from mouse spinal cord, a compartment of the central nervous system that is significantly affected in neuromuscular diseases such as amyotrophic lateral sclerosis. This method relies on differential centrifugation to separate the mitochondria from the other subcellular compartments.

Keywords: Mitochondria(线粒体), Isolation(隔离), CNS(中枢神经系统), ALS(肌萎缩性脊髓侧索硬化症), Neurodegeneration(神经退行性疾病)

Materials and Reagents

  1. Isoflurane (Isosol) (Vedco, catalog number: 50201 )
  2. Fatty acid (FA) free bovine serum albumin (Sigma-Aldrich, catalog number: 85041C )
  3. cOmpleteTM ULTRA protease inhibitor tablets, EDTA-free (Roche, catalog number: 0589253001 )
  4. OptiPrepTM density gradient medium (Sigma-Aldrich, catalog number: D1556 )
  5. Bradford reagent (Bio-Rad, catalog number: 500-0001 )
  6. Mannitol (Sigma-Aldrich, catalog number: M4125 )
  7. Phosphate buffer saline (PBS) (see Recipes)
  8. 1 M Tris-HCl (see Recipes)
  9. 0.5 M EDTA (see Recipes)
  10. Buffer M (see Recipes)
  11. Buffer M + 0.45% FA free BSA + protease inhibitors (see Recipes)
  12. Buffer M + protease inhibitors (see Recipes)

Equipment

  1. 10 ml disposable plastic syringe fitted (heat sealed) with a 200 μl pipette tip
    Using a Bunsen burner, heat and partially melt the wide end of a 200 μl pipette tip. Before the plastic has time to harden quickly insert the melted end of the 200 μl pipette tip into the luer-lok of a disposable plastic 10 ml syringe. Let the assembly cool and check the tightness of the seal by running 10 ml of water at high pressure through the syringe/tip assembly.  
  2. Sharp heavy dissection duty scissors
  3. Euthanasia jar
  4. Balance and plastic weight boat
  5. Tweezers
  6. 1 ml glass homogenizer with loose and tight pestles (Kimble Chase Kontes) (Fisher Scientific, catalog number: 885300-0001 )
  7. Table top refrigerated centrifuge
  8. Ultrafuge
  9. Thinwall, Ultra-ClearTM 5 ml 13 x 51 mm ultrafuge tubes (Beckman Coulter, catalog number: 344057 )
  10. SW55Ti swing-out rotor (Beckman Coulter, model: 342194 )
  11. Pipettes
  12. 50 ml conical

Procedure

  1. Preparation
    1. Put the following items on ice:
      1. Homogenizer + pestles (those should be placed in a 50 ml conical on ice).
      2. 50 ml of Buffer M + 0.45% BSA + cOmpleteTM.
      3. 50 ml of Buffer M + cOmpleteTM.
    2. Transfer the following items to the cold room:
      All the items placed on ice.
      1. Pipettes
      2. Pipette tips
      3. Tweezers
      4. Small bucket of water to rinse pestles
      5. Swing out buckets for SW55Ti
      6. UltraClear SW55Ti tubes
      7. OptiPrep
    3. Set up the animal dissection area with:
      1. Euthanasia chamber with Isoflurane
      2. Heavy duty scissors
      3. 10 ml syringe filled with PBS
      4. Small plastic weigh trays placed on ice
      5. Tweezers

  2. The start of procedure

    Figure 1. Procedure for purification of mitochondria from mouse spinal cord using differential centrifugation

    1. Terminally anesthetize the mouse using isoflurane in the euthanasia chamber.
    2. Cervicaly dislocate the animal, decapitate and section the spinal column at the iliac crest (just above the hips). Flush the spinal cord from the spinal column using the 10 ml syringe filled with PBS. Insert the tip at the caudal opening of the spinal column and eject about 5 ml of PBS. This should flush out the spinal cord from the rostral end of the severed spinal column. If a clean flushing of the spinal cord cannot be achieved, section the dorsal column below the front limbs and repeat the flushing procedure. In this case make sure to save the piece of spinal cord located within the small piece of spinal column resulting from the cut below the front-limbs. Place the spinal cord, after removing excess PBS, in the small plastic weigh trays placed on ice.
    3. Weigh the spinal cord rapidly and place it back on ice.
    4. Steps B5 to B18 are all performed in the cold room.
    5. While working in the cold room, transfer the spinal cord with cold tweezers in the 1 ml glass homogenizer on ice. Add 10 volumes of buffer M + 0.45% FA free BSA + cOmpleteTM (e.g. if the spinal cord weighs 60 mg add 600 μl of buffer).
    6. Gently homogenize the tissue on ice with 10-15 strokes (just enough to completely dissociate the tissue) using the cooled loose pestle. Avoid forming air bubbles.
      Note: Generating bubbles during the homogenization process can lead to disruption of intracellular membranes and denaturing of proteins, both of which should be avoided to reduce damage to the mitochondria.
    7. Gently Homogenize the tissue on ice with 15 strokes using the tight pestle. Avoid forming air bubbles.
    8. Centrifuge at 1,000 x g for 5 min at 4 °C and transfer the supernatant (S1) to a fresh tube on ice.
      Note:The 1,000 x g supernatant generated at this step is composed mostly of cytosol with heavy and light membranes. The pellet contains mostly unbroken cells and nuclei.
    9. Resuspend the pellet (P1) in 10 volumes of buffer M + 0.45% FA free BSA + cOmpleteTM and gently homogenize 15x using the tight pestle. Avoid forming air bubbles.
      Note:This step is used to further release cytosol and intracellular organelles from the remaining unbroken cells.
    10. Centrifuge at 1,000 x g for 5 min at 4 °C and pool the resulting supernatant (S1) with the supernatant (S2) from the step 8.
    11. Centrifuge the pooled supernatants at 1,000 x g for 5 min at 4 °C.
      Note: This step ensures that any contaminating unbroken cells or nuclei are removed from the cytosol/intracellular membrane subcellular fraction.
    12. Collect supernatant (S3) staying clear of the pellet and spin it at 12,000 x g for 10 min at 4 °C.
    13. Prepare Beckman UltraClearTM tubes with 200 μl of OptiPrepTM and 600 μl of Buffer M + 0.45% FA free BSA + cOmpleteTM. Mix well and store on ice.
    14. Discard the supernatant (S4) resulting from step B12 and gently (but thoroughly) resuspend the pellet (P4) using in 150 μl of buffer M + 0.45% FA free BSA + cOmpleteTM.
      Note:The 12,000 x g supernatant generated at this step is composed mostly of cytosol with light membranes. The pellet contains mostly heavy intracellular membranes (mitochondria).
    15. Once the pellet is completely resuspended transfer all of it (recording the final volume) into the Beckman UltraClearTM tubes prepared in step B13. Complement with buffer M + 0.45% FA free BSA + cOmpleteTM to make a final volume of 1 ml and mix gently (but thoroughly) with a 1 ml pipette.
    16. Transfer the UltraClearTM tubes to the SW55Ti swing out buckets on ice and centrifuge the tubes in a SW55Ti rotor using a high speed centrifuge at 17,000 x g (equivalent to 13,400 rpm) at 4 °C for 17 minutes (remove breaks).
      Note: The heavy membrane fraction is centrifuged in an OptiPrep gradient to remove most of the contaminants (for example myelin and endoplasmic reticulum) from the heavy membrane preparation. The UltraClearTM tubes can be reused if washed well after use (without detergent) and stored dry.
    17. Gently remove the tubes from the buckets and place them on ice. Using gentle aspiration remove supernatant including the top white layer.
    18. Gently resuspend the pellet (P5) in 1 ml of buffer M + cOmpleteTM (without completely dispersing it) and centrifuge at 12,000 x g for 10 minutes at 4 °C.
      Note: After the OptiPrep gradient the heavy membranes are resuspended in buffer M without BSA so as to wash off the BSA from the previous steps to more accurately measure the protein concentration in step B20.
    19. Resuspend the pellet (P6) in 10-20 μl of buffer M + cOmpleteTM by gently flicking the tube with your fingers and placing back on ice as quickly as possible. Make sure pellet is completely resuspended before performing the protein quantification.
      Note: It is important to resuspend the heavy membranes at a high concentration since this reduces damage to the organelles. Resuspending the organelles by flicking also reduces organelle damage.
    20. Estimate protein quantity using a Bradford assay on 1 μl of resuspended pellet. Once the protein concentration is determined, add a final concentration of 0.45% FA free BSA to the suspension using a 4.5% FA free BSA stock to avoid diluting the preparation.
      Note: FA free BSA is added back to the heavy membrane preparation after protein quantification to allow for better preservation of mitochondrial activity by reducing uncoupling of the organelle. Ideally mitochondrial activity (oxygen consumption) should be measured on a fraction of the preparation to determine the quality of the organelle and the damage that may have been sustained during preparation.

Recipes

  1. PBS (1 L)
    Start with 800 ml of distilled water
    Add 8 g of NaCl
    Add 0.2 g of KCl
    Add 1.44 g of Na2HPO4
    Add 0.24 g of KH2PO4
    Adjust the pH to 7.4 with HCl
    Add distilled water to a total volume of 1 L
  2. 1 M Tris-HCl, pH 7.4 (500 ml)
    Mix 60.57 g of Tris base with 400 ml of dH2O
    When Tris base is dissolved pH to 7.4 using concentrated HCl
    Complete to 500 ml with dH2O
    Stored at 4 °C
  3. 0.5 M EDTA, pH 8.0 (500 ml)
    Mix 73.06 g of EDTA with 400 ml of dH2O
    Adjust pH to 8.0 using concentrated KOH
    Complete to 500 ml with dH2O
    Stored at 4 °C
  4. Buffer M (500 ml)
    Mix 19.1 g  of mannitol into 400 ml of dH2O
    Add 11.9 g of sucrose
    Add 5 ml of 1 M Tris-HCl pH 7.4
    Add 1 ml of 0.5 M EDTA pH 8.0
    Complete to 500 ml with dH2O
    Filter sterilize (0.22 μm)
    Stored at 4 °C
    Note: Before the start of the isolation procedure prepare Recipes 5 and 6.
  5. Buffer M + 0.45% FA free BSA + protease inhibitors
    50 ml Buffer M + 0.225 g FA free BSA + 1 tablet of cOmpleteTM protease inhibitors (stored at 4 °C)
  6. Buffer M + protease inhibitors    
    50 ml Buffer M + 1 tablet of cOmpleteTM protease inhibitors (stored at 4 °C)

Acknowledgments

This protocol is adapted from Parone et al. (2013) and Vande Velde et al. (2008).

References

  1. Parone, P. A., Da Cruz, S., Han, J. S., McAlonis-Downes, M., Vetto, A. P., Lee, S. K., Tseng, E. and Cleveland, D. W. (2013). Enhancing mitochondrial calcium buffering capacity reduces aggregation of misfolded SOD1 and motor neuron cell death without extending survival in mouse models of inherited amyotrophic lateral sclerosis. J Neurosci 33(11): 4657-4671.    
  2. Vande Velde, C., Miller, T. M., Cashman, N. R. and Cleveland, D. W. (2008). Selective association of misfolded ALS-linked mutant SOD1 with the cytoplasmic face of mitochondria. Proc Natl Acad Sci U S A 105(10): 4022-4027.   

简介

线粒体是在几种细胞过程中发挥关键作用的真核细胞器,包括能量产生,脂肪酸的β-氧化和钙稳态的调节。在过去20年中,线粒体研究发现线粒体是程序性细胞死亡过程的核心,线粒体功能障碍涉及许多疾病,包括广泛的神经系统疾病如帕金森病,阿尔茨海默病,亨廷顿病和肌萎缩性侧索硬化。为了识别和研究与特定神经病学条件相关的线粒体功能的变化,线粒体通常从疾病中最受影响的中枢神经系统的隔室中分离出来。在这里,我们描述了从小鼠脊髓分离线粒体的方案,中枢神经系统的一个隔室在神经肌肉疾病如肌萎缩性侧索硬化症中受到显着影响。该方法依赖于差速离心法将线粒体与其他亚细胞隔离区分离。

关键字:线粒体, 隔离, 中枢神经系统, 肌萎缩性脊髓侧索硬化症, 神经退行性疾病

材料和试剂

  1. 异氟烷(Isosol)(Vedco,目录号:50201)
  2. 不含脂肪酸(FA)的牛血清白蛋白(Sigma-Aldrich,目录号:85041C)
  3. cOmplete TM ULTRA蛋白酶抑制剂片剂,无EDTA(Roche,目录号:0589253001)
  4. OptiPrep TM密度梯度介质(Sigma-Aldrich,目录号:D1556)
  5. Bradford试剂(Bio-Rad,目录号:500-0001)
  6. 甘露醇(Sigma-Aldrich,目录号:M4125)
  7. 磷酸盐缓冲盐水(PBS)(见配方)
  8. 1 M Tris-HCl(参见配方)
  9. 0.5 M EDTA(见配方)
  10. 缓冲区M(参见配方)
  11. 缓冲液M + 0.45%FA游离BSA +蛋白酶抑制剂(参见配方)
  12. 缓冲液M +蛋白酶抑制剂(参见配方)

设备

  1. 10毫升一次性塑料注射器(热密封)用200微升吸头
    使用本生灯,加热并部分熔化200μl移液管尖端的宽端。 在塑料有时间硬化之前,快速地将200μl移液管尖端的熔化端插入一次性塑料10ml注射器的鲁尔管。 让组件冷却,并通过注射器/尖端组件在高压下运行10ml水来检查密封的紧密性。  
  2. 夏普重型夹层责任剪刀
  3. 安乐死瓶
  4. 平衡和塑料重物船
  5. 镊子
  6. 1ml具有松散和紧密杵的玻璃匀浆器(Kimble Chase Kontes)(Fisher Scientific,目录号:885300-0001)
  7. 台式冷冻离心机
  8. Ultrafuge
  9. Thinwall,Ultra-Clear TM 5ml 13×51mm超滤管(Beckman Coulter,目录号:344057)
  10. SW55Ti转出转子(Beckman Coulter,型号:342194)
  11. 移液器
  12. 50ml圆锥形

程序

  1. 制备
    1. 将以下物品放在冰上:
      1. 均化器+杵(那些应放置在冰上的50ml锥形)
      2. 50ml缓冲液M + 0.45%BSA + cOmplete TM
      3. 50ml缓冲液M + cOmplete TM
    2. 将以下物品转移到冷藏室:
      所有的项目放在冰上。
      1. 移液器
      2. 移液器提示
      3. 镊子
      4. 小桶水冲洗杵
      5. 为SW55Ti摆出水桶
      6. UltraClear SW55Ti管
      7. OptiPrep
    3. 设置动物解剖区域:
      1. 安乐死室与异氟烷
      2. 重型剪刀
      3. 10ml装有PBS的注射器
      4. 小型塑料称重托盘放在冰上
      5. 镊子

  2. 程序开始

    图1.使用差速离心从小鼠脊髓中纯化线粒体的程序

    1. 在安乐死室中使用异氟烷终止麻醉小鼠。
    2. Cervicaly脱臼动物,断头并在髂嵴(恰好在臀部上方)切断脊柱。使用装有PBS的10ml注射器从脊柱冲洗脊髓。插入尖端在脊柱的尾部开口,并弹出约5毫升的PBS。这将从被切断的脊柱的嘴端冲出脊髓。如果无法实现脊髓的清洁冲洗,请将前柱下方的背柱分段,并重复冲洗程序。在这种情况下,请确保保存脊髓小块中脊髓导致的前肢下方切割片。在除去多余的PBS后,将脊髓置于置于冰上的小塑料称重盘中
    3. 快速称重脊髓,将其放回冰上。
    4. 步骤B5到B18都在寒冷的房间里进行。
    5. 在冷室中工作时,用冰镊在1ml玻璃匀浆器中在冰上转移脊髓。加入10体积的缓冲液M + 0.45%FA游离BSA + cOmplete TM sup (例如,如果脊髓重60mg,加入600μl缓冲液)。
    6. 使用冷却的松散杵,用10-15次敲击轻轻匀浆冰上的组织(刚好足以完全解离组织)。避免形成气泡。
      注意:在匀浆过程中产生气泡可导致细胞内膜破裂和蛋白质变性,应避免这两种情况,以减少对线粒体的损害。
    7. 使用紧杵在15次敲击下轻轻匀浆冰上的组织。避免形成气泡。
    8. 在4℃下1,000×g离心5分钟,并将上清液(S1)转移到冰上的新管中。
      注意:在此步骤产生的1,000×g上清液主要由具有重和轻膜的胞质溶胶组成。颗粒含有大部分未破裂的细胞和核。
    9. 将沉淀(P1)重悬在10体积的缓冲液M + 0.45%FA游离BSA + cOmplete TM中,并使用紧杵轻轻匀化15x。避免形成气泡。
      注意:此步骤用于从剩余的未破裂细胞中进一步释放细胞质和细胞内细胞器。
    10. 在4℃下1,000×g离心5分钟,并将所得上清液(S1)与来自步骤8的上清液(S2)混合。
    11. 在4℃下以1,000×g离心混合的上清液5分钟。
      注意:此步骤确保任何污染的未破坏的细胞或细胞核从细胞质/细胞内膜亚细胞部分去除。
    12. 收集上清液(S3),保持离开沉淀并在4℃下以12,000xg旋转10分钟。
    13. 用200μlOptiPrep TM和600μl缓冲液M + 0.45%FA游离BSA + cOmplete TM制备Beckman UltraClear TM 管。混合好,存放在冰上。
    14. 弃去从步骤B12得到的上清液(S4),并使用在150μl缓冲液M + 0.45%FA游离BSA + cOmplete TM缓冲液中轻轻(但彻底)重悬沉淀(P4)。
      注意:在此步骤产生的12,000xg上清液主要由具有光膜的细胞溶质组成。沉淀物主要包含重的细胞内膜(线粒体)。
    15. 一旦沉淀物完全重悬,将其全部(记录最终体积)转移到在步骤B13中制备的Beckman UltraClear TM管中。用缓冲液M + 0.45%无游离BSA + cOmplete TM补充以使终体积为1ml,并用1ml移液管轻轻(但彻底)混合。
    16. 将UltraClear TM 管转移到SW55Ti在冰上摆动的桶中,并使用高速离心机以17,000×g(相当于13,400rpm)在SW55Ti转子中离心管4℃17分钟(除去断裂)。
      注意:将重膜级分在OptiPrep梯度中离心以从重膜制备物中除去大部分污染物(例如髓磷脂和内质网)。 UltraClear TM 管可以重复使用,如果在使用后清洗(没有洗涤剂),并保存干燥。
    17. 轻轻地从水桶中取出管,将它们放在冰上。使用温和抽吸除去上清液,包括顶部白色层。
    18. 轻轻地将沉淀(P5)重悬在1ml缓冲液M + cOmplete TM(未完全分散)中,并在4℃以12,000×g离心10分钟。
      注意:在OptiPrep梯度后,将重膜悬浮在不含BSA的缓冲液M中,以便洗去前述步骤中的BSA,以更准确地测量步骤B20中的蛋白质浓度。
    19. 通过用你的手指轻轻地轻拂管,并尽快放回冰上,重悬在10-20微升缓冲液M + cOmplete TM 中的沉淀(P6)。在进行蛋白质定量之前,确保沉淀物完全重悬 注意:重要的是以高浓度重悬浮重膜,因为这减少对细胞器的损伤。通过轻弹重悬细胞器还可以减少细胞器损伤。
    20. 使用Bradford测定法对1μl重悬浮的沉淀估计蛋白质量。一旦确定蛋白质浓度,使用4.5%FA游离BSA原液将最终浓度为0.45%无游离BSA添加到悬浮液中,以避免稀释制剂。
      注意:在蛋白质定量后,将无游离的BSA加回到重膜制备物中,以允许通过减少细胞器的解偶联来更好地保持线粒体活性。理想地,线粒体活性(氧消耗)应当在制剂的一部分上测量,以确定细胞器的质量和在制备过程中可能持续的损伤。

食谱

  1. PBS(1 L)
    开始用800毫升蒸馏水
    加入8克NaCl
    加入0.2g KCl
    加入1.44g Na 2 HPO 4
    加入0.24g KH 2 PO 4 sub/
    用HCl
    调节pH至7.4 加入蒸馏水至总体积为1 L
  2. 1M Tris-HCl,pH7.4(500ml) 将60.57g的Tris碱与400ml的dH 2 O混合 当Tris碱用浓HCl溶解pH至7.4时 用dH 2 O完全达到500ml
    储存在4°C
  3. 0.5M EDTA,pH8.0(500ml) 将73.06g EDTA与400ml dH 2 O混合 使用浓KOH将pH调节至8.0 用dH 2 O完全达到500ml
    储存在4°C
  4. 缓冲液M(500ml)
    混合19.1 g 的甘露醇加入到400ml的dH 2 O中 加入11.9g蔗糖
    加入5ml的1M Tris-HCl pH7.4 加入1ml 0.5M EDTA pH 8.0/v 用dH 2 O完全达到500ml
    过滤灭菌(0.22μm)
    储存在4°C
    注意: 隔离程序之前,请先准备食谱5和6。
  5. 缓冲液M + 0.45%FA游离BSA +蛋白酶抑制剂 50ml缓冲液M + 0.225g FA游离BSA + 1片cOmplete TM蛋白酶抑制剂(在4℃下储存)
  6. 缓冲液M +蛋白酶抑制剂   
    50ml缓冲液M + 1片cOmplete TM蛋白酶抑制剂(在4℃下储存)

致谢

该协议改编自Parone等人(2013)和Vande Velde等人(2008)。

参考文献

  1. Parone,P.A.,Da Cruz,S.,Han,J.S.,McAlonis-Downes,M.,Vetto,A.P.,Lee,S.K.,Tseng,E.and Cleveland,D.W。(2013)。 增强线粒体钙缓冲能力可减少错误折叠的SOD1和运动神经元细胞死亡的聚集,而不会延长小鼠模型的生存期 的遗传性肌萎缩性侧索硬化。 J Neurosci 33(11):4657-4671。  
  2. Vande Velde,C.,Miller,T.M.,Cashman,N.R.and Cleveland,D.W。(2008)。 错折叠的ALS连锁突变型SOD1与线粒体细胞质面的选择性关联。 em> Proc Natl Acad Sci USA 105(10):4022-4027。   
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Copyright: © 2013 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. Parone, P. A., Cruz, S. D. and Cleveland, D. W. (2013). Mitochondrial Isolation and Purification from Mouse Spinal Cord. Bio-protocol 3(21): e961. DOI: 10.21769/BioProtoc.961.
  2. Parone, P. A., Da Cruz, S., Han, J. S., McAlonis-Downes, M., Vetto, A. P., Lee, S. K., Tseng, E. and Cleveland, D. W. (2013). Enhancing mitochondrial calcium buffering capacity reduces aggregation of misfolded SOD1 and motor neuron cell death without extending survival in mouse models of inherited amyotrophic lateral sclerosis. J Neurosci 33(11): 4657-4671.    
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