这项研究是由美国国家心脏，肺和血液研究所资助HL-103542，并在散麻醉研究基金会的杰出开拓者奖CAP支持。

准备<ol><li>制备的1M K-HEPES溶液（pH调节至7.2，用KOH）。 </li><li>制备0.5M的K-EGTA溶液（调节pH值至8.0，用KOH）。 </li><li>制备的1M KH 2 PO 4的储备溶液。 </li><li>准备1米氯化镁储备溶液。 </li><li>制备均质缓冲液（pH 7.2）中300mM的蔗糖，10mM的K-HEPES，和1mM K-EGTA。保存在4℃。 </li><li>制备呼吸缓冲液250mM的蔗糖，2mM的KH 2 PO 4，10mM的MgCl 2的，20mM的K-HEPES缓冲液（pH值7.2）和0.5mM的K-EGTA（pH 8.0）中。保存在4℃。 </li><li>制备10倍的PBS原液溶解80克的NaCl，2克的KCl，14.4称量Na 2 HPO 4，和2.4克KH 2 PO 4加入到1 L双蒸H 2 O的 （pH 7.4）中。 </li><li>吹打百毫升10X PBS为1升双蒸H 2 O准备1X PBS </li><li>准备枯草杆菌蛋白酶A股称重在4毫克枯草杆菌蛋白酶A的以1.5 ml离心管中。储存在-20℃直到使用。 </li><li>准备BSA股票称取20毫克的BSA为1.5 ml离心管中。储存在-20℃直到使用。 </li></ol>线粒体的分离（图1） <ol><li>紧接隔离，溶解枯草杆菌蛋白酶A在1毫升均质缓冲液。 </li><li>紧接隔离，BSA溶解在1毫升均质缓冲液。 </li><li>使用6毫米活检样本冲和存储在1X PBS在冰50毫升离心管中收集两个新鲜组织标本。 </li><li>转移组织两个6毫米拳含有5ml冰冷的均质缓冲液的分离Ç管。 </li><li>由嵌合在组织离解的离解Ç管均质化的组织，并选择预先设定的线粒体隔离周期（60秒均化）。 </li><li>拆下分离ç管冰桶。 </li><li>加入250μl的枯草杆菌蛋白酶原液的匀浆，通过倒置混合，并培育在冰上匀浆10分钟。 </li><li>放置一个40微米的筛网过滤器上的冰和预湿的过滤器以均质缓冲液50ml锥形离心管中，并过滤匀浆成冰的50ml锥形离心管中。 </li><li>加入250微升新鲜制备的BSA储备液，向滤液并通过倒置混合。 注:省略这一步，如果需要线粒体蛋白的决心。 </li><li>放置一个40微米的过滤器上的冰和预湿的过滤器以均质缓冲液50ml锥形离心管中，并过滤匀浆成冰的50ml锥形离心管中。 </li><li>放置一个10微米的过滤器上的冰和预湿的过滤器以均质缓冲液50ml锥形离心管中，并过滤匀浆成冰的50ml锥形离心管中。 </li><li>将滤液转移到2预冷1.5毫升离心管和离心9000 xg离心10分钟4ºC。 </li><li>除去上清液并重新悬浮和结合粒料在1ml冰冷的呼吸缓冲液。 </li></ol> ATP含量注意:要确定分离线粒体的ATP荧光检测可以用一个ATP检测试剂盒进行的代谢活动。的协议，试剂和标准的测定试剂盒提供。的流程的概要进行说明。 <ol><li>平衡试剂盒至室温。 </li><li>准备10毫摩尔ATP原液在1170微升双蒸水溶解冻干ATP的颗粒。储存的ATP冰上标准储备液，准备线粒体样本。 </li><li>加入5毫升底物缓冲溶液，以冻干底物液的小瓶中。轻轻摇匀后放置在黑暗中。 </li><li>加入100μl呼吸缓冲液至黑色，不透明的底部，96孔板的所有孔中。 </li><li>从所制备的样品中添加10微升线粒体向每个孔ø发黑色，不透明的底部，96孔板中。注:样品被镀以一式三份。包括行的标准，三口井为阴性对照组（呼吸缓冲器）。 </li><li>加入50微升的哺乳动物细胞溶解液到所有孔中，包括标准和控制。 </li><li>孵育板在37℃5分钟，在定轨摇床上以125转每分钟。 </li><li>在孵育制备的0.1毫米，0.05毫米，0.01毫米，0.005毫米，0.001毫米，和0.0001毫摩尔ATP从10毫摩尔ATP储备溶液浓度的ATP标准。商店的标准在冰上。 </li><li>温育后，加入10微升的三磷酸腺苷标准，以相应的孔中的板图上标明。注意:标准进行双份。 </li><li>加入50微升重组的底物溶液的各孔中。 </li><li>孵育板在37℃在定轨振荡器进行5分钟，在125转每分钟。 </li><li>打开GEN5 1.11软件与分光光度计相连的计算机上。 </li><li>在"创建新项目"，点击"实验"。 </li><li>点击"默认协议"。单击"确定"。 </li><li>在左侧列中，选择"协议"，然后选择"程序"。 </li><li>选择"延迟"。设置为00:10:00。单击"确定"。 </li><li>选择"读取"。从下拉菜单中选择"发光"。调整其他设置如下:读取类型的端点，积分时间0:01.00 MM:SS.SS，过滤器设置1，发射孔，光学部位顶部，感光度100，顶探头垂直偏移1.00毫米。 </li><li>当板已准备好进行分析，点击最上面一排的"阅读板块"图标。点击"读取"。当分光光度计打开后，将板与A1孔在左上角的托盘。随着温度的对话框将打开。点击"读取"。注:值越高，关联与增加ATP水平和较高的代谢活动。 </li></ol>

<ol>
	<li>van Loo, G., Saelens, X., van Gurp, M., MacFarlane, M., Martin, S. J., Vandenabeele, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+role+of+mitochondrial+factors+in+apoptosis:+a+Russian+roulette+with+more+than+one+bullet.">The role of mitochondrial factors in apoptosis: a Russian roulette with more than one bullet.</a> Cell Death Differ. 9 (10), 1031-1042 (2002).</li><li>Szabadkai, G., Duchen, M. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mitochondria:+the+hub+of+cellular+Ca2++signaling.">Mitochondria: the hub of cellular Ca2+ signaling.</a> Physiology (Bethesda). 23, 84-94 (2008).</li><li>Chan, D. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mitochondria:+Dynamic+Organelles+in+Disease,+Aging,+and+Development.">Mitochondria: Dynamic Organelles in Disease, Aging, and Development.</a> Cell. 125 (7), 1241-1252 (2006).</li><li>Picard, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mitochondrial+structure+and+function+are+disrupted+by+standard+Isolation+methods.">Mitochondrial structure and function are disrupted by standard Isolation methods.</a> PLoS ONE. 6 (3), e18317 (2011).</li><li>Bensley, R. R., Hoerr, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Studies+on+cell+structure+by+freeze-drying+method;+preparation+and+properties+of+mitochondria.">Studies on cell structure by freeze-drying method; preparation and properties of mitochondria.</a> Anat Rec. 60, 449-455 (1934).</li><li>Claude, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fractionation+of+mammalian+liver+cells+by+differential+centrifugation+II.+Experimental+procedures+and+results.">Fractionation of mammalian liver cells by differential centrifugation II. Experimental procedures and results.</a> J Exp Med. 84 (1), 61-89 (1946).</li><li>Hogeboom, H., Schneider, W. C., Palade, G. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cytochemical+studies+of+mammalian+tissues;+isolation+of+intact+mitochondria+from+rat+liver;+some+biochemical+properties+of+mitochondria+and+submicroscopic+particulate+material.">Cytochemical studies of mammalian tissues; isolation of intact mitochondria from rat liver; some biochemical properties of mitochondria and submicroscopic particulate material.</a> J Biol Chem. 172 (2), 619-635 (1948).</li><li>Ernster, L., Schtaz, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mitochondria:+a+historical+Review.">Mitochondria: a historical Review.</a> J Cell Biol. 91 (3 Pt 2), 227s-255s (1981).</li><li>Pallotti, F., Lenaz, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+and+subfractionation+of+mitochondria+from+animal+cells+and+tissue+culture+lines.">Isolation and subfractionation of mitochondria from animal cells and tissue culture lines.</a> Methods Cell Biol. 80, 3-44 (2007).</li><li>Schmitt, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+semi-automated+method+for+isolating+functionally+intact+mitochondria+from+cultured+cells+and+tissue+biopsies.">A semi-automated method for isolating functionally intact mitochondria from cultured cells and tissue biopsies.</a> Anal Biochem. 443 (1), 66-74 (2013).</li><li>Fern&#225;ndez-Vizarra, E., Ferr&#237;n, G., P&#233;rez-Martos, A., Fern&#225;ndez-Silva, P., Zeviani, M., Enr&#237;quez, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+of+mitochondria+for+biogenetical+studies:+An+update.">Isolation of mitochondria for biogenetical studies: An update.</a> Mitochondrion. 10 (3), 253-262 (2010).</li><li>Graham, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chapter+3,+Unit+3.3,+Isolation+of+mitochondria+from+tissues+and+cells+by+differential+centrifugation.">Chapter 3, Unit 3.3, Isolation of mitochondria from tissues and cells by differential centrifugation.</a> Curr Protoc Cell Biol. , (2001).</li><li>Frezza, C., Cipolat, S., Scorrano, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Organelle+isolation:+functional+mitochondria+from+mouse+liver,+muscle+and+cultured+fibroblasts.">Organelle isolation: functional mitochondria from mouse liver, muscle and cultured fibroblasts.</a> Nat Protoc. 2 (2), 287-295 (2007).</li><li>Wieckowski, M. R., Giorgi, C., Lebiedzinska, M., Duszynski, J., Pinton, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+of+mitochondria-associated+membranes+and+mitochondria+from+animal+tissues+and+cells.">Isolation of mitochondria-associated membranes and mitochondria from animal tissues and cells.</a> Nat Protoc. 4 (11), 1582-1590 (2009).</li><li>Gostimskaya, I., Galkin, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preparation+of+highly+coupled+rat+heart+mitochondria.">Preparation of highly coupled rat heart mitochondria.</a> J Vis Exp. (43), (2010).</li><li>Gross, V. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+of+functional+mitochondria+from+rat+kidney+and+skeletal+muscle+without+manual+homogenization.">Isolation of functional mitochondria from rat kidney and skeletal muscle without manual homogenization.</a> Anal Biochem. 418 (2), 213-223 (2011).</li><li>Masuzawa, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transplantation+of+autologously+derived+mitochondria+protects+the+heart+from+ischemia-reperfusion+injury.">Transplantation of autologously derived mitochondria protects the heart from ischemia-reperfusion injury.</a> American J Physiol Heart and Circ Physiol. 304 (7), (2013).</li><li>McCully, J. D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Injection+of+isolated+mitochondria+during+early+reperfusion+for+cardioprotection.">Injection of isolated mitochondria during early reperfusion for cardioprotection.</a> American J Physiol Heart and Circ Physiol. 296 (1), 94-105 (2009).</li><li>Olson, M. S., Von Korff, R. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Changes+in+endogenous+substrates+of+isolated+rabbit+heart+mitochondria+during+storage.">Changes in endogenous substrates of isolated rabbit heart mitochondria during storage.</a> J Biol Chem. 242 (2), 325-332 (1967).</li><li>Diepart, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+of+methods+for+measuring+oxygen+consumption+in+tumor+cells+in+vitro.">Comparison of methods for measuring oxygen consumption in tumor cells in vitro.</a> Anal Biochem. 396 (2), 250-256 (2010).</li><li>Lanza, I. R., Nair, K. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+assessment+of+isolated+mitochondria+in+vitro.">Functional assessment of isolated mitochondria in vitro.</a> Methods Enzymol. 457, 349-372 (2009).</li></ol>

所有的动物都按照现行体制的指导方针进行处理。我们没有竞争的经济利益披露。

成功地分离出使用该协议，必须保持所有的解决方案和组织样品在冰上保持线粒体活力线粒体。在冰上保持甚至当，分 ​​离线粒体将表现出随着时间的推移19的功能活性的降低。我们建议所有的解决方案和补充预先做好准备。我们预先称重并存储枯草杆菌蛋白酶A在4毫克等分试样在1.5ml微量离心管中，并将其存储在-20℃。同样的BSA被预先称重并存储在在1.5ml微量离心管20毫克等分，可以储...

A correction was made to: <a href="http://www.jove.com/video/51682">Rapid Isolation And Purification Of Mitochondria For Transplantation By Tissue Dissociation And Differential Filtration.</a> An optional step was added to step 7 of the &#39;Mitochondrial Isolation&#39; section of the protocol. The step is:
1. Optional in the case that the tissue is fibrous: Centrifuge the solution at 750 x g for 4 min.

A correction was made to: Rapid Isolation And Purification Of Mitochondria For Transplantation By Tissue Dissociation And Differential Filtration.

Erratum: Rapid Isolation And Purification Of Mitochondria For Transplantation By Tissue Dissociation And Differential Filtration

存在的每一个细胞在体内除了红血细胞中的线粒体，并参与了大量重要的细胞代谢过程1-4。由于这些诸多功能，线粒体损伤可能具有不利的影响3。为了研究线粒体功能和功能障碍的若干线粒体的分离方法进行了描述。线粒体隔离日至1940年5-8最早公布的帐目。第一次记录尝试通过研磨肝组织在研钵中，然后离心以盐溶液在低速5,8证实线粒体隔离。后来，其他群体扩大后，原来的程序，证明组织分离的基础上差速离心6-8。这些早期方法形成的电流的技术，往往掺入均质化，和/或差速离心9-15的基础。 homogenizati数并离心步骤不同协议之间。这些重复的步骤，增加了时间线粒体隔离，并最终降低的可行性。此外，手动均化可引起线粒体损伤和不一致的结果，如果没有适当的控制，10,16。 最近，我们使用的同质化和差速离心分离线粒体移植到心肌组织17,18。这个冗长分离过程需要约90分钟，该方法的临床适用性因此受到限制。要允许在临床和手术治疗急性治疗用途，我们已经开发出可以在不到30分钟内完成快速的线粒体分离过程。 该协议的主要好处是标准化的组织分解允许统一和组织的一致同质化是不容易用手工同质化来实现的。在乳房清洁度离子，代替差速离心的使用微分过滤消除了耗时的和重复离心步骤，允许对高度纯化的，可行的和呼吸能力的线粒体的更快速分离。 隔离可行和呼吸主管线粒体在不到30分钟的能力允许临床应用。这种隔离协议有潜力的冠状动脉旁路移植术（CABG）和其他治疗程序中使用。

<table><tbody><tr><td>Sucrose</td><td>Sigma Aldrich</td><td>84100</td><td></td></tr><tr><td>HEPES</td><td>Sigma Aldrich</td><td>H4034</td><td></td></tr><tr><td>EGTA</td><td>Sigma Aldrich</td><td>E4378</td><td></td></tr><tr><td>Substilsin A</td><td>Sigma Aldrich</td><td>P5380</td><td></td></tr><tr><td>BSA</td><td>Sigma Aldrich</td><td>A7906</td><td></td></tr><tr><td>KH&lt;sub&gt;2&lt;/sub&gt;PO&lt;sub&gt;4&lt;/sub&gt;</td><td>Sigma Aldrich</td><td>P5379</td><td></td></tr><tr><td>MgCl&lt;sub&gt;2&lt;/sub&gt;</td><td>Sigma Aldrich</td><td>M8266</td><td></td></tr><tr><td>NaCl</td><td>Sigma Aldrich</td><td>S6191</td><td></td></tr><tr><td>KCl</td><td>Fisher Scientific</td><td>P2173</td><td></td></tr><tr><td>Na&lt;sub&gt;2&lt;/sub&gt;HPO&lt;sub&gt;4&lt;/sub&gt;</td><td>Fisher Scientific</td><td>S374</td><td></td></tr><tr><td>ATPlite Luminescence Assay System,&lt;br/&gt; 1,000 Assay Kit</td><td>Perkin Elmer</td><td>6016941</td><td></td></tr><tr><td>50 ml Conical Tubes</td><td>BD</td><td>352098</td><td></td></tr><tr><td>40 &amp;mu;m Nylon Filters</td><td>BD&amp;nbsp;</td><td>352340</td><td></td></tr><tr><td>GentleMACS C tube</td><td>Miltenyl Biotech</td><td>120-005-331</td><td></td></tr><tr><td>1.5 ml Eppendorf tube</td><td>Fisher Scientific</td><td>05-408-129</td><td></td></tr><tr><td>6 mm biopsy punch</td><td>Miltex</td><td>33-36</td><td></td></tr><tr><td>10 &amp;mu;m Pluristrainer</td><td>Pluriselect</td><td>43-500-10-03</td><td></td></tr><tr><td>Eppendorf Centrifuge 5415C</td><td>Marshall Scientific</td><td>EP-5415C&amp;nbsp;</td><td></td></tr><tr><td>GentleMACS Dissociator&amp;nbsp;</td><td>Miltenyl Biotech</td><td>130-093-235</td><td></td></tr><tr><td>96-well plates, tissue culture treated</td><td>VWR</td><td>82050-732</td><td></td></tr><tr><td>Rotomax 120 orbital shaker</td><td>Heidolph</td><td>544-41200-00</td><td></td></tr><tr><td>Synergy H4 Hybrid Multi-Mode&lt;br/&gt; &amp;nbsp;Microplate Reader</td><td>BioTek</td><td></td><td></td></tr><tr><td>Multisizer 4 Coulter Counter</td><td>Beckman Coulter</td><td>A63076</td><td></td></tr><tr><td>Oxytherm System</td><td>Hansatech Instruments</td><td></td><td></td></tr><tr><td>Hemacytometer</td><td>Fisher Scientific</td><td>267110</td><td></td></tr></tbody></table>

rapid isolation and purification of mitochondria for transplantation by tissue dissociation and differential filtration

使用差速离心和/或聚蔗糖梯度离心之前描述的线粒体分离方法需要60至100分钟内完成。我们描述了线粒体利用商业组织离解和鉴别过滤哺乳动物的活组织切片检查的快速提取方法。在这个协议中，手动均化置换为组织离解的标准化的均匀化周期。这使得均匀和组织的一致均质化是不容易用手工匀化来实现的。以下组织离解，在匀浆通过尼龙网过滤器，它消除重复离心步骤过滤。其结果是，线粒体隔离可以在不到30分钟来进行。此隔离协议产生约2×10 10活和呼吸从0.18±0.04克（湿重）的组织样品主管线粒体。

一个数字，概述采用组织分离和鉴别过滤线粒体的隔离程序步骤如图1。总过程时间小于30分钟。 组织样品用6毫米活检穿孔获得。组织的重量为0.18±0.04克（湿重）。线粒体分离为通过粒径数量确定的数目为2.4×10 10±0.1×10 10线粒体对骨骼肌和2.75×10 10±0.1×10 10线粒体肝制剂（ 图2A）。以允许比较线粒体数目也通过血细胞计数器确定。线?...

Watch this Scientific Journal Video about Rapid Isolation And Purification Of Mitochondria For Transplantation By Tissue Dissociation And Differential Filtration at JoVE.com

Rapid Isolation And Purification Of Mitochondria For Transplantation By Tissue Dissociation And Differential Filtration

一种用于线粒体的从哺乳动物组织活检快速分离方法进行说明。大鼠肝或骨骼肌肉制剂匀浆与商业组织离解和线粒体是通过尼龙网过滤分离差过滤。线粒体隔离时间是相对于60 &lt;30分钟 - 用替代的方法100分钟。

快速分离纯化线粒体移植用组织分离和鉴别过滤

Previously described mitochondrial isolation methods using differential centrifugation and/or Ficoll gradient centrifugation require 60 to 100 min to ...

rapid-isolation-purification-mitochondria-for-transplantation-tissue

Research

JoVE Journal

Biology

38.0K 次观看.  Beth Israel Deaconess Medical Center and Harvard Medical School. 一种用于线粒体的从哺乳动物组织活检快速分离方法进行说明。大鼠肝或骨骼肌肉制剂匀浆与商业组织离解和线粒体是通过尼龙网过滤分离差过滤。线粒体隔离时间是相对于60 

视频: Rapid Isolation And Purification Of Mitochondria For Transplantation By Tissue Dissociation And Differential Filtration

Rapid Isolation of the Mitoribosome from HEK Cells

The human mitochondria possess a dedicated set of ribosomes (mitoribosomes) that translate 13 essential protein components of the oxidative phosphorylation complexes encoded by the mitochondrial genome. Since all proteins synthesized by human mitoribosomes are integral membrane proteins, human mitoribosomes are tethered to the mitochondrial inner membrane during translation. Compared to the cytosolic ribosome the mitoribosome has a sedimentation coefficient of 55S, half the rRNA content, no 5S rRNA and 36 additional proteins. Therefore, a higher protein-to-RNA ratio and an atypical structure make the human mitoribosome substantially distinct from its cytosolic counterpart.
Despite the central importance of the mitoribosome to life, no protocols were available to purify the intact complex from human cell lines. Traditionally, mitoribosomes were isolated from mitochondria-rich animal tissues that required kilograms of starting material. We reasoned that mitochondria in dividing HEK293-derived human cells grown in nutrient-rich expression medium would have an active mitochondrial translation, and, therefore, could be a suitable source of material for the structural and biochemical studies of the mitoribosome. To investigate its structure, we developed a protocol for large-scale purification of intact mitoribosomes from HEK cells. Herein, we introduce nitrogen cavitation method as a faster, less labor-intensive and more efficient alternative to traditional mechanical shear-based methods for cell lysis. This resulted in preparations of the mitoribosome that allowed for its structural determination to high resolution, revealing the composition of the intact human mitoribosome and its assembly intermediates. Here, we follow up on this work and present an optimized and more cost-effective method requiring only ~1010 cultured HEK cells. The method can be employed to purify human mitoribosomal translating complexes, mutants, quality control assemblies and mitoribosomal subunits intermediates. The purification can be linearly scaled up tenfold if needed, and also applied to other types of cells.

Mitochondria have specialized ribosomes that diverged from their bacterial and cytoplasmic counterparts. Here we show how mitoribosomes can be obtained from their native compartment in HEK cells. The method involves isolation of mitochondria from suspension cells and consequent purification of mitoribosomes.

HEK 细胞 Mitoribosome 的快速分离

The human mitochondria possess a dedicated set of ribosomes (mitoribosomes) that translate 13 essential protein components of the oxidative ...

科研

生物化学

Preparation and Respirometric Assessment of Mitochondria Isolated from Skeletal Muscle Tissue Obtained by Percutaneous Needle Biopsy

Respirometric profiling of isolated mitochondria is commonly used to investigate electron transport chain function. We describe a method for obtaining samples of human Vastus lateralis, isolating mitochondria from minimal amounts of skeletal muscle tissue, and plate based respirometric profiling using an extracellular flux (XF) analyzer. Comparison of respirometric profiles obtained using 1.0, 2.5 and 5.0 &#956;g of mitochondria indicate that 1.0 &#956;g is sufficient to measure respiration and that 5.0 &#956;g provides most consistent results based on comparison of standard errors. Western blot analysis of isolated mitochondria for mitochondrial marker COX IV and non-mitochondrial tissue marker GAPDH indicate that there is limited non-mitochondrial contamination using this protocol. The ability to study mitochondrial respirometry in as little as 20 mg of muscle tissue allows users to utilize individual biopsies for multiple study endpoints in clinical research projects.

Methods for biopsy of Vastus lateralis, preparation of purified mitochondria, and respirometric profiling are described. The use of small muscle volume makes this technique suitable for clinical research applications.

线粒体的制备及透气性评估，从骨骼肌组织通过穿刺活检获得的分离

Respirometric profiling of isolated mitochondria is commonly used to investigate electron transport chain function. We describe a method for obtaining ...

医学

分离线粒体接触位点

An Improved Method to Isolate Mitochondrial Contact Sites

Mitochondria are present in virtually all eukaryotic cells and perform essential functions that go far beyond energy production, for instance, the synthesis of iron-sulfur clusters, lipids, or proteins, Ca2+ buffering, and the induction of apoptosis. Likewise, mitochondrial dysfunction results in severe human diseases such as cancer, diabetes, and neurodegeneration. In order to perform these functions, mitochondria have to communicate with the rest of the cell across their envelope, which consists of two membranes. Therefore, these two membranes have to interact constantly. Proteinaceous contact sites between the mitochondrial inner and outer membranes are essential in this respect. So far, several contact sites have been identified. In the method described here, Saccharomyces cerevisiae mitochondria are used to isolate contact sites and, thus, identify candidates that qualify for contact site proteins. We used this method to identify the mitochondrial contact site and cristae organizing system (MICOS) complex, one of the major contact site-forming complexes in the mitochondrial inner membrane, which is conserved from yeast to humans. Recently, we further improved this method to identify a novel contact site consisting of Cqd1 and the Por1-Om14 complex.

作者聚焦:揭开线粒体接触位点和架构见解 

Mitochondrial contact sites are protein complexes that interact with mitochondrial inner and outer membrane proteins. These sites are essential for the communication between the mitochondrial membranes and, thus, between the cytosol and the mitochondrial matrix. Here, we describe a method to identify candidates qualifying for this specific class of proteins.

一种分离线粒体接触位点的改进方法

Mitochondria are present in virtually all eukaryotic cells and perform essential functions that go far beyond energy production, for instance, the ...

Preparation of Highly Coupled Rat Heart Mitochondria

The function of mitochondria in generation of cellular ATP in the process of oxidative phosphorylation is widely recognised. During the past decades there have been significant advances in our understanding of the functions of mitochondria other than the generation of energy. These include their role in apoptosis, acting as signalling organelles, mammalian development and ageing as well as their contribution to the coordination between cell metabolism and cell proliferation. Our understanding of biological processes modulated by mitochondria is based on robust methods for isolation and handling of intact mitochondria from tissues of the laboratory animals. Mitochondria from rat heart is one of the most common preparations for past and current studies of cellular metabolism including studies on knock-out animals.
Here we describe a detailed rapid method for isolation of intact mitochondria with a high degree of coupling. Such preparation of rat heart mitochondria is an excellent object for functional and structural research on cellular bioenergetics, transport of biomolecules, proteomic studies and analysis of mitochondrial DNA, proteins and lipids.

We describe &#1072; protocol for isolation of pure, highly coupled rat heart mitochondria for functional or structural studies of cellular bioenergetics, biophysical measurements, proteomics or mitochondrial DNA and lipids analysis.

高度耦合的大鼠心肌线粒体的制备

The function of mitochondria in generation of cellular ATP in the process of oxidative phosphorylation is widely recognised. During the past decades there ...

生物学

用于超复合物分析的小组织样品中的线粒体分离

Isolation of Mitochondria for Mitochondrial Supercomplex Analysis from Small Tissue and Cell Culture Samples

Over the last decades, the evidence accumulated about the existence of respiratory supercomplexes (SCs) has changed our understanding of the mitochondrial electron transport chain organization, giving rise to the proposal of the "plasticity model." This model postulates the coexistence of different proportions of SCs and complexes depending on the tissue or the cellular metabolic status. The dynamic nature of the assembly in SCs would allow cells to optimize the use of available fuels and the efficiency of electron transfer, minimizing reactive oxygen species generation and favoring the ability of cells to adapt to environmental changes. 
More recently, abnormalities in SC assembly have been reported in different diseases such as neurodegenerative disorders (Alzheimer's and Parkinson's disease), Barth Syndrome, Leigh syndrome, or cancer. The role of SC assembly alterations in disease progression still needs to be confirmed. Nevertheless, the availability of enough amounts of samples to determine the SC assembly status is often a challenge. This happens with biopsy or tissue samples that are small or have to be divided for multiple analyses, with cell cultures that have slow growth or come from microfluidic devices, with some primary cultures or rare cells, or when the effect of particular costly treatments has to be analyzed (with nanoparticles, very expensive compounds, etc.). In these cases, an efficient and easy-to-apply method is required. This paper presents a method adapted to obtain enriched mitochondrial fractions from small amounts of cells or tissues to analyze the structure and function of mitochondrial SCs by native electrophoresis followed by in-gel activity assays or western blot.

作者聚焦：揭示氧化磷酸化系统动力学和线粒体在健康和疾病中的作用

This protocol describes a technique for the analysis of respiratory supercomplexes when only small amounts of samples are available.

从小组织和细胞培养样品中分离线粒体用于线粒体超复合物分析 

Over the last decades, the evidence accumulated about the existence of respiratory supercomplexes (SCs) has changed our understanding of the mitochondrial ...

<meta charset="utf8"></head><body>从啮齿动物中分离线粒体以进行高质量的生物能量研究

Robust Mitochondrial Isolation from Rodent Cardiac Tissue

Mitochondrial isolation has been practiced for decades, following procedures established by pioneers in the fields of molecular biology and biochemistry to study metabolic impairments and disease. Consistent mitochondrial quality is necessary to properly investigate mitochondrial physiology and bioenergetics; however, many different published isolation methods are available for researchers. Although different experimental strategies require different isolation methods, the basic principles and procedures are similar. This protocol details a method capable of extracting well-coupled mitochondria from a variety of tissue sources, including small animals and cells. The steps outlined include organ dissection, mitochondrial purification, protein quantification, and various quality control checks. The primary quality control metric used to identify high-quality mitochondria is the respiratory control ratio (RCR). The RCR is the ratio of the respiratory rate during oxidative phosphorylation to the rate in the absence of ADP. Alternative metrics are discussed. While high RCR values relative to their tissue source are obtained using this protocol, several steps can be optimized to suit the individual needs of researchers. This procedure is robust and has consistently resulted in isolated mitochondria with above-average RCR values across animal models and tissue sources.

<meta charset="utf8"></head><body>作者聚焦:揭示线粒体磷酸钙在心力衰竭和生物能量学中的作用

Bioenergetic and metabolomic studies on mitochondria have revealed their multifaceted role in many diseases, but the isolation methods for these organelles vary. The method detailed here is capable of purifying high-quality mitochondria from multiple tissue sources. Quality is determined by respiratory control ratios and other metrics assessed with high-resolution respirometry.

从啮齿动物心脏组织中稳健地分离线粒体 

Mitochondrial isolation has been practiced for decades, following procedures established by pioneers in the fields of molecular biology and biochemistry ...

快速分离纯化线粒体移植用组织分离和鉴别过滤

摘要

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此视频中的章节

HEK 细胞 Mitoribosome 的快速分离

线粒体的制备及透气性评估，从骨骼肌组织通过穿刺活检获得的分离

一种分离线粒体接触位点的改进方法

一种分离线粒体接触位点的改进方法

一种分离线粒体接触位点的改进方法

高度耦合的大鼠心肌线粒体的制备

从小组织和细胞培养样品中分离线粒体用于线粒体超复合物分析

从小组织和细胞培养样品中分离线粒体用于线粒体超复合物分析

从小组织和细胞培养样品中分离线粒体用于线粒体超复合物分析

从啮齿动物心脏组织中稳健地分离线粒体