This research was supported in part by NSF grant CHE-1229562 (VDGM), the Office of Undergraduate Research at Elon University, the Elon Chemistry Department, and the Elon Lumen Prize (TL and TAD), the Elon College Fellows Program (JAC), and the Elon College Honors Program (TAD).

所有的动物实验符合卫生指引国家机构，并批准了维克森林大学的动物护理和使用委员会。对种鸽的SOD1G93A [B6SJL-TGN（SOD1-G93A）1Gur]小鼠模型是从杰克逊实验室（巴尔港，ME）获得。非转基因的野生型（WT）的女性和男性的SOD1G93A [B6SJL-TGN（SOD1-G93A）1Gur]饲养生成SOD1G93A小鼠，并在实验中使用的非转基因野生型同窝小鼠。 1.模型研究<ol><li>细胞培养<ol><li>在5％CO 2维持人胚肾细胞（HEK-T293）细胞在DMEM补充有10％热灭活的胎牛血清，1％青霉素-链霉素和1％L-谷氨酰胺，在37℃下。 </li><li>细胞生长在T75烧瓶，并让他们达到90％汇合。 </li><li>进行通过胰蛋白酶消化（0.25％胰蛋白酶-EDTA）3-5分钟细胞分裂，在37℃在5％CO 2 </sub&gt;，胰蛋白酶与5分钟，于700×g离心在4℃下等体积细胞培养基，和离心的细胞失活。 </li><li>吸出介质，重悬在培养基中的细胞沉淀。 </li><li>种子细胞在适当的烧瓶或板7×10 4个细胞到T75烧瓶，以细胞因子治疗之前48小时。这应该给〜70％，密度在治疗时间。 </li><li> 24小时后通过吸移的媒体，并与无血清培养基的相同体积（DMEM，无菌）替换它血清饥饿的细胞。 </li><li>制备TNF-α，IL1-β，和IFN-γ，从冻干粉末用超纯水以5微克/微升，10毫微克/微升的工作浓度，和10毫微克/微升，分别和它们添加到孔/瓶。 </li><li>治疗血清通过直接对适当的烧瓶中的细胞培养基中加入可溶的细胞因子饥饿细胞。治疗包括个体细胞因子，所有3鸡尾酒（简称为"* 3"），或STERILE水作为对照（被称为"0"）。 </li><li>评估和收获前培养处理的细胞24,48或72小时。 </li></ol></li><li>细胞活力评估<ol><li>使5毫克/毫升的溶液的3-（4,5-二甲基吡啶-2-基）-2,5-二苯基溴化（MTT）在1×PBS中。 </li><li>对于12孔板，加入100微升的MTT溶液，以每孔，轻轻摇动，以确保均匀分布。 </li><li>在5％CO 2孵育板15分钟，1小时，在37℃，并在显微镜对紫色着色的存在下进行检查。如果没有紫色的存在，再次漩涡，让细胞在5分钟的间隔孵化，直到紫色观察。的必要时间量应当由每个研究者决定。 </li><li>使用重复移液器，加入700微升的MTT溶剂（4mM的HCl和0.1％NP40中异丙醇）的每个孔中，摇动板（S），在室温下搅拌5-10分钟，或直到所有紫色已离开细胞。如果紫颜色不出来的细胞，增加一个额外的200微升溶剂，继续漩涡。 </li><li>进行分析，以从每个100微升井，并放置到96孔板中并读取酶标仪使用570纳米和630纳米的参考波长，但595nm处也是可以接受的，只要读数采取一致的设置。 </li></ol></li><li> ALS的动物模型<ol><li>所有的动物实验符合卫生指引国家机构，并批准了维克森林大学的动物护理和使用委员会。对种鸽的SOD1G93A [B6SJL-TGN（SOD1-G93A）1Gur]小鼠模型是从杰克逊实验室（巴尔港，ME）获得。非转基因的野生型（WT）的女性和男性的SOD1G93A [B6SJL-TGN（SOD1-G93A）1Gur]饲养生成SOD1G93A小鼠，并在实验中使用的非转基因野生型同窝小鼠。 </li><li>与对SOD1突变25标准引物进行基因分型。 </li></ol></li></ol> 2.隔离线粒体注:重要的是要迅速开展工作，并保留一切冰上整个过程中是非常重要的。 <ol><li>线粒体隔离缓冲器（MIB），脊髓隔离缓冲液（SC，MIB）和实验缓冲液制剂（EB）的<ol><li>提前时间MIB和EB准备以下的股票解决方案。 </li><li>使的1M Tris / MOPS通过在70毫升的H 2 O中溶解12.1克Tris碱的加入干MOPS得到pH值至7.4。音量调节至100毫升决赛。过滤消毒，并储存于4℃。 </li><li>使1M的Kphos通过混合80.2毫升K 2 HPO 4和19.8毫升KH 2 PO 4的。室温保存。 </li><li>使0.2M的EGTA /的Tris加入3.8克EGTA至10中的H 2 O毫升添加的1M Tris / MOPS直到溶解，30-40〜毫升。调整到50毫升，无菌过滤并储存在室温下。需要注意的是pH值将是〜6.7。 </li><li>使1M的谷氨酸通过加入10ml1M溶液谷氨酸。过滤消毒和储存4℃。 </li><li>使1M的苹果酸通过加入10ml苹果酸的1​​M溶液。添加的Tris / MOPS至50毫米。过滤消毒，并储存于4℃。 </li><li>使MIB的在200毫蔗糖，10毫摩尔Tris / MOPS，pH值7.4，和1mM EGTA /的Tris的浓度。过滤消毒，并储存于4℃。 </li><li>使SC的MIB在250毫蔗糖，20mM的HEPES-KOH pH为7.5，10mM的氯化钾，1.5mM的氯化镁 ，1mM EDTA中，1毫EGTA，1mM的DTT和蛋白酶抑制剂混合物的浓度。 </li><li>使电子束在125毫米氯化钾的浓度，10毫摩尔Tris / MOPS，pH为7.4，5mM的谷氨酸，2.5mM的苹果酸盐，1mM的磷酸钾，pH 7.4中，和10mM EGTA /的Tris。过滤消毒，并储存于4℃。 </li></ol></li><li>之前收获细胞设备的准备。 <ol><li>冲洗的小玻璃容器和同质化杵三次，用无菌水，并放置冰上。 </li><li>收集必要的物品，如标准钻，细胞涉农供应链个人，将1.5ml，15毫升和50毫升试管和解决方案。 </li></ol></li><li>线粒体隔离<ol><li>培养细胞<ol><li>对于每个样品类型中，使用细胞的2 T175烧瓶中。 </li><li>确保细胞是〜90％汇合，并在对照实验中，或板使用和处理如在步骤1.2，如上所述。 </li><li>置于工作台顶部，抽吸培养基，用每次15毫升1×PBS中洗两次的贴壁细胞。 </li><li>吸出缓冲液和刮烧瓶从烧瓶底部去除粘附细胞。 </li><li>添加15毫升1×PBS中，以每个烧瓶，漩涡，并转移至个别的15毫升锥形管中并置于冰上。 </li><li>一旦刮削操作后，离心管5分钟，于700×g离心，4℃下使用使用吊桶式转子台式离心机。 </li><li>吸取上清液，重悬在1ml MIB的每个颗粒。 </li><li>同时结合了悬浮液和转移到一个小的玻璃容器内的同质化。 </li><li>添加MIB到容器中，直至到达缓冲器的第一行。均质化细胞附连到钻头以中速3道次的杵。确保该容器是在冰上在此步骤中，不以除去液体上面的杵和使用一个稳定的速度连续通行证。 </li><li>转移均质溶液到50毫升锥形管中。 </li><li>使用18号1半英寸针吸取溶液到3毫升注射器，用27号半英寸针驱逐它放回锥形管在冰上。照顾驱逐靠在管子的内壁上的溶液，作为利用该力对细胞膜的破坏。 </li><li>重复注射器的步骤，共5次。 </li><li>在使用水平转头台式离心机将溶液转移入15ml锥形管中，并离心5分钟，在600×g离心，4℃。 </li><li>小心取出上清液，在三个1.5毫升管分配。 注:Mitochondria是在该第一，低速旋后的上清，而细胞膜和不间断的细胞沉淀。 </li><li>离心管在一个固定角转子以10,000×g离心，4℃5分钟。 </li><li>抽吸上清液，并结合所述球粒在100微升的MIB，并立即放置冰上。 注:线粒体是在这种高速旋转后的沉淀。线粒体通常将保持其膜电势为〜2-3离子分离后冰并作为浓缩储备溶液在MIB中最稳定的。 </li></ol></li><li>从小鼠组织中线粒体隔离<ol><li>根据IACUC协议麻醉的小鼠。在5.0％设置一个蒸发器以诱导麻醉。证实该动物是由于缺乏反射脚捏或眨眼反射的麻醉当眼睛接近或轻拍用棉签。保持在麻醉状态下对整个手术过程的小鼠通过保持在1.5％和2.0％的蒸发器。 </li><li> EXCISE在冰冷的1X PBS肝脏和脊髓从每个动物和地点另行 - / - ，以洗去任何血液。 </li><li>对肝脏，将组织转移至称量盘上的冰和剁成细碎片用新鲜刀片1分钟。 </li><li>加组织和适当的缓冲液（MIB用于肝脏或SC的MIB用于脊髓）的容器中，直至到达缓冲器的第一行。同质化组织与杵用手五关。确保该容器是在冰上在此步骤中，不以除去液体上面的杵和使用一个稳定的速度连续通行证。 </li><li>转移匀浆清洁15ml试管。 </li><li>离心管在一个固定角转子，在750×g离心，4℃，10分钟。 </li><li>保存上清液进入洁净管，将它们放在冰上。 注:线粒体是在该第一，低速旋后上清，而细胞膜和不间断的细胞沉淀。 </li><li>重新暂停脊髓粒料我ñ500微升SC MIB的。 </li><li>再均质各三次，只填充容器的一半与SC MIB这段时间。 </li><li>传输新匀浆至新管。 </li><li>离心机的在一个固定的角​​度转子以750×g离心，4℃，10分钟。 </li><li>结合这些新的上清液，其含有较多的线粒体，从每个样本的第一上清液。 </li><li>离心管在一个固定角转子以10,000×g离心，4℃5分钟。 </li><li>抽吸上清液和重悬肝粒料在500μl的MIB和在50μl的SC MIB中的脊髓粒料并立即放在冰上。 注:线粒体是在这种高速旋转后的沉淀。线粒体通常将保持其膜电势为〜2-3离子分离后冰并作为浓缩储备溶液在MIB中最稳定的。 </li><li>执行一个蛋白质浓度测定以估计在溶液线粒体的浓度。按照指令S表示使用市售蛋白质测定试剂盒或类似的方法26。典型线粒体的浓度如下:2 T175烧瓶收率〜2毫克/毫升，1小鼠肝脏〜3-5毫克/毫升，以及1小鼠脊髓〜1-3毫克/毫升。 </li></ol></li></ol></li><li>使用研发大鼠/小鼠细胞色素C的Quantikine ELISA试剂盒（改编自由制造商提供的协议）细胞色素c的测定。 <ol><li>之前立即设置的反应中，稀线粒体至0.5mg / ml的工作浓度与EB和在1.5ml管中用于反应（通常为30-50微升每管线粒体的）分发稀释线粒体。 </li><li>添加任EB或DMSO，在总体积的1％，到稀释的线粒体和孵化在台式反应7-10分钟。这些反应是稳定长达30分钟，在室温下，如果需要较长的时间帧。 </li><li>通过离心在一个固定角转子以10,000×g离心，4℃下进行5分钟，并小心粒料线粒体LY分离上清液并将其放置每一个单独1.5毫升管中。 注:管既可以在-20℃冷冻后用于分析或立即使用。 </li><li>通过比较，在沉淀和上清液27细胞色素C的浓度测定细胞色素C的释放。 <ol><li>通过增溶粒料中有0.5％的Tx-100在1×PBS中的反应的原体积制备样品。 </li><li>对于每个样品，准备2个孔上的ELISA板，一个用于现在溶解沉淀，一个用于从通过加入100微升的细胞色素c的共轭反应的上清液（直使用选自瓶）加100微升0.5％的TX- 100在1×PBS中，然后所有的粒料或上清液样品。 </li><li>轻轻涡旋盘低设置（2-4级），持续20秒混合，盖，孵育1小时，37℃。 </li><li>接着，根据厂家专业洗涤平板4次，洗涤缓冲液稀释呃的说明。通过点击该井的纸巾取出多余的解决方案。 </li><li>接着，添加150微升的1:1的A + B显影液向每个孔，孵育该板在室温下20-30分钟，在黑暗中。 </li><li>最后，添加50微升的终止溶液至每孔，并采取吸光度读数在540nm处用酶标仪。通过比较，在沉淀与上清液每个反应的细胞色素C的量计算细胞色素c释放的量。 </li></ol></li></ol></li></ol>线粒体（不典型增生）功能3.评估<ol><li>紧接之前的反应都设置，稀释线粒体至0.5mg / ml的工作浓度与EB和放置在1.5ml管中用于反应（通常为30-50微升线粒体每管被使用）。 </li><li>线粒体治疗<ol><li>添加适当的治疗（EB控制，1μMFCCP与50纳米缬氨霉素混合，10μM鱼藤酮，为5μM寡，2mM的KCN，或200μM的ADP，终浓度），以在1/10分离反应次的稀释线粒体的总体积。孵育在台式反应7分钟。 注:通过皮肤处理这些物质用作误食或吸收时可能是有害的应当小心。 </li><li>稀TMRE，重新溶于无菌水中以100微米的工作浓度，并储存在-20℃下，向2μM和添加量等于线粒体反应体积。 </li><li>孵育在台式反应7分钟。 </li><li>通过离心在一个固定角转子以10,000×g离心，4℃5分钟粒料线粒体。 </li><li>每个样品的上清液体积的负荷一半到396孔板和读取荧光。 注:TMRE的激发和发射波长应根据使用体积和板，其将用于制造商的说明进行优化化验。使用标准的384孔黑色板用25微升1μM的TMRE（最终反应浓度）得到最强的信号用的激发/发射波长&#160; 485纳米/ 535纳米。 </li><li>计算在对照（EB）和各处理通过分割从板阅读器，用于通过从对照样品的RFU的试验样品获得的相对荧光值（RFU）之间的荧光的折叠的差异。 </li></ol></li></ol>

<ol>
	<li>Madeira, V. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Overview+of+mitochondrial+bioenergetics.">Overview of mitochondrial bioenergetics.</a> Methods In Molecular Biology. 810, 1-6 (2012).</li><li>Sakamuru, 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=Application+of+a+homogenous+membrane+potential+assay+to+assess+mitochondrial+function.">Application of a homogenous membrane potential assay to assess mitochondrial function.</a> Physiological Genomics. 44, 495-503 (2012).</li><li>Clayton, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structure+and+function+of+the+mitochondrial+genome.">Structure and function of the mitochondrial genome.</a> Journal Of Inherited Metabolic Disease. 15, 439-447 (1992).</li><li>Schatz, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+protein+import+system+of+mitochondria.">The protein import system of mitochondria.</a> The Journal of Biological Chemistry. 271, 31763-31766 (1996).</li><li>Mayer, A., Neupert, W., Lill, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mitochondrial+protein+import:+reversible+binding+of+the+presequence+at+the+trans+side+of+the+outer+membrane+drives+partial+translocation+and+unfolding.">Mitochondrial protein import: reversible binding of the presequence at the trans side of the outer membrane drives partial translocation and unfolding.</a> Cell. 80, 127-137 (1995).</li><li>Green, D. R., Kroemer, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+pathophysiology+of+mitochondrial+cell+death.">The pathophysiology of mitochondrial cell death.</a> Science. 305, 626-629 (2004).</li><li>Shamas-Din, A., Kale, J., Leber, B., Andrews, D. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanisms+of+action+of+Bcl-2+family+proteins.">Mechanisms of action of Bcl-2 family proteins.</a> Cold Spring Harbor Perspectives In Biology. 5, a008714 (2013).</li><li>Hardwick, J. M., Youle, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=SnapShot:+BCL-2+proteins.">SnapShot: BCL-2 proteins.</a> Cell. 138, 404 (2009).</li><li>Zoratti, M., Szabo, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+mitochondrial+permeability+transition.">The mitochondrial permeability transition.</a> Biochimica et Biophysica Acta. 1241, 139-176 (1995).</li><li>Wu, C. C., Bratton, S. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulation+of+the+intrinsic+apoptosis+pathway+by+reactive+oxygen+species.">Regulation of the intrinsic apoptosis pathway by reactive oxygen species.</a> Antioxidants &amp; Redox Signaling. 19, 546-558 (2013).</li><li>Kuwana, T., 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=BH3+domains+of+BH3-only+proteins+differentially+regulate+Bax-mediated+mitochondrial+membrane+permeabilization+both+directly+and+indirectly.">BH3 domains of BH3-only proteins differentially regulate Bax-mediated mitochondrial membrane permeabilization both directly and indirectly.</a> Molecular Cell. 17, 525-535 (2005).</li><li>Joshi, D. C., Bakowska, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Determination+of+mitochondrial+membrane+potential+and+reactive+oxygen+species+in+live+rat+cortical+neurons.">Determination of mitochondrial membrane potential and reactive oxygen species in live rat cortical neurons.</a> Journal Of Visualized Experiments. , (2011).</li><li>Vinsant, 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=Characterization+of+early+pathogenesis+in+the+SOD1(G93A)+mouse+model+of+ALS:+part+I,+background+and+methods.">Characterization of early pathogenesis in the SOD1(G93A) mouse model of ALS: part I, background and methods.</a> Brain and Behavior. 3, 335-350 (2013).</li><li>Vinsant, 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=Characterization+of+early+pathogenesis+in+the+SOD1(G93A)+mouse+model+of+ALS:+part+II,+results+and+discussion.">Characterization of early pathogenesis in the SOD1(G93A) mouse model of ALS: part II, results and discussion.</a> Brain and Behavior. 3, 431-457 (2013).</li><li>Rajapakse, N., Shimizu, K., Payne, M., Busija, D. <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+characterization+of+intact+mitochondria+from+neonatal+rat+brain.">Isolation and characterization of intact mitochondria from neonatal rat brain.</a> Brain research. Brain Research Protocols. 8, 176-183 (2001).</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> Analytical Biochemistry. 443, 66-74 (2013).</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> Nature Protocols. 2, 287-295 (2007).</li><li>Debatin, K. M., Poncet, D., Kroemer, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chemotherapy:+targeting+the+mitochondrial+cell+death+pathway.">Chemotherapy: targeting the mitochondrial cell death pathway.</a> Oncogene. 21, 8786-8803 (2002).</li><li>Del Gaizo Moore, V., 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=Chronic+lymphocytic+leukemia+requires+BCL2+to+sequester+prodeath+BIM,+explaining+sensitivity+to+BCL2+antagonist+ABT-737.">Chronic lymphocytic leukemia requires BCL2 to sequester prodeath BIM, explaining sensitivity to BCL2 antagonist ABT-737.</a> The Journal of Clinical Investigation. 117, 112-121 (2007).</li><li>Del Gaizo Moore, V., Schlis, K. D., Sallan, S. E., Armstrong, S. A., Letai, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=BCL-2+dependence+and+ABT-737+sensitivity+in+acute+lymphoblastic+leukemia.">BCL-2 dependence and ABT-737 sensitivity in acute lymphoblastic leukemia.</a> Blood. 111, 2300-2309 (2008).</li><li>Li, N., 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+complex+I+inhibitor+rotenone+induces+apoptosis+through+enhancing+mitochondrial+reactive+oxygen+species+production.">Mitochondrial complex I inhibitor rotenone induces apoptosis through enhancing mitochondrial reactive oxygen species production.</a> The Journal of Biological Chemistry. 278, 8516-8525 (2003).</li><li>Hynes, J., 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=Investigation+of+drug-induced+mitochondrial+toxicity+using+fluorescence-based+oxygen-sensitive+probes.">Investigation of drug-induced mitochondrial toxicity using fluorescence-based oxygen-sensitive probes.</a> Toxicological Sciences : An Official Journal Of The Society of Toxicology. 92, 186-200 (2006).</li><li>Papkovsky, D. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Methods+in+optical+oxygen+sensing:+protocols+and+critical+analyses.">Methods in optical oxygen sensing: protocols and critical analyses.</a> Methods in enzymology. 381, 715-735 (2004).</li><li>Galluzzi, L., 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=Methods+for+the+assessment+of+mitochondrial+membrane+permeabilization+in+apoptosis.">Methods for the assessment of mitochondrial membrane permeabilization in apoptosis.</a> Apoptosis : An International Journal On Programmed Cell Death. 12, 803-813 (2007).</li><li>Gurney, M. E., 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=Motor+neuron+degeneration+in+mice+that+express+a+human+Cu,Zn+superoxide+dismutase+mutation.">Motor neuron degeneration in mice that express a human Cu,Zn superoxide dismutase mutation.</a> Science. 264, 1772-1775 (1994).</li><li>Smith, P. K., 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=Measurement+of+protein+using+bicinchoninic+acid.">Measurement of protein using bicinchoninic acid.</a> Analytical Biochemistry. 150, 76-85 (1985).</li><li>Certo, 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=Mitochondria+primed+by+death+signals+determine+cellular+addiction+to+antiapoptotic+BCL-2+family+members.">Mitochondria primed by death signals determine cellular addiction to antiapoptotic BCL-2 family members.</a> Cancer Cell. 9, 351-365 (2006).</li><li>Hogeboom, G. H., Schneider, W. C., Pallade, 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> The Journal Of Biological Chemistry. 172, 619-635 (1948).</li><li>Hogeboom, G. H., Claude, A., Hotch-Kiss, R. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+distribution+of+cytochrome+oxidase+and+succinoxidase+in+the+cytoplasm+of+the+mammalian+liver+cell.">The distribution of cytochrome oxidase and succinoxidase in the cytoplasm of the mammalian liver cell.</a> The Journal of Biological Chemistry. 165, 615-629 (1946).</li><li>Marques-Fernandez, F., 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=TNFalpha+induces+survival+through+the+FLIP-L-dependent+activation+of+the+MAPK/ERK+pathway.">TNFalpha induces survival through the FLIP-L-dependent activation of the MAPK/ERK pathway.</a> Cell Death &amp; Disease. 4, e493 (2013).</li><li>Chao, C. C., Hu, S., Ehrlich, L., Peterson, P. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Interleukin-1+and+tumor+necrosis+factor-alpha+synergistically+mediate+neurotoxicity:+involvement+of+nitric+oxide+and+of+N-methyl-D-aspartate+receptors.">Interleukin-1 and tumor necrosis factor-alpha synergistically mediate neurotoxicity: involvement of nitric oxide and of N-methyl-D-aspartate receptors.</a> Brain, Behavior, And Immunity. 9, 355-365 (1995).</li><li>Downen, M., Amaral, T. D., Hua, L. L., Zhao, M. L., Lee, S. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neuronal+death+in+cytokine-activated+primary+human+brain+cell+culture:+role+of+tumor+necrosis+factor-alpha.">Neuronal death in cytokine-activated primary human brain cell culture: role of tumor necrosis factor-alpha.</a> Glia. 28, 114-127 (1999).</li><li>Schildberger, A., Rossmanith, E., Weber, V., Falkenhagen, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Monitoring+of+endothelial+cell+activation+in+experimental+sepsis+with+a+two-step+cell+culture+model.">Monitoring of endothelial cell activation in experimental sepsis with a two-step cell culture model.</a> Innate Immunity. 16, 278-287 (2010).</li><li>Brown, M. R., 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=Nitrogen+disruption+of+synaptoneurosomes:+an+alternative+method+to+isolate+brain+mitochondria.">Nitrogen disruption of synaptoneurosomes: an alternative method to isolate brain mitochondria.</a> Journal Of Neuroscience Methods. 137, 299-303 (2004).</li><li>Wong, A., Cortopassi, G. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-throughput+measurement+of+mitochondrial+membrane+potential+in+a+neural+cell+line+using+a+fluorescence+plate+reader.">High-throughput measurement of mitochondrial membrane potential in a neural cell line using a fluorescence plate reader.</a> Biochemical and Biophysical Research Communications. 298, 750-754 (2002).</li><li>Blattner, J. R., He, L., Lemasters, J. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Screening+assays+for+the+mitochondrial+permeability+transition+using+a+fluorescence+multiwell+plate+reader.">Screening assays for the mitochondrial permeability transition using a fluorescence multiwell plate reader.</a> Analytical Biochemistry. 295, 220-226 (2001).</li><li>Kataoka, M., Fukura, Y., Shinohara, Y., Baba, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analysis+of+mitochondrial+membrane+potential+in+the+cells+by+microchip+flow+cytometry.">Analysis of mitochondrial membrane potential in the cells by microchip flow cytometry.</a> Electrophoresis. 26, 3025-3031 (2005).</li><li>Del Gaizo Moore, V., Payne, R. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transactivator+of+transcription+fusion+protein+transduction+causes+membrane+inversion.">Transactivator of transcription fusion protein transduction causes membrane inversion.</a> The Journal of Biological Chemistry. 279, 32541-32544 (2004).</li></ol>

The authors have nothing to disclose.

HEK-293T细胞用重组细胞因子的治疗导致适量的细胞死亡超过48小时（ 图1）。诱导的TNF-α处理的细胞死亡的量是类似于以前报道的研究30和多种细胞因子是大于总结性的量与任何细胞因子的共施用后细胞活力降低单独也与文献31,32一致。调整数量和类型的细胞因子治疗，以及鸡尾酒单个细胞因子比较的能力，使学习对肾脏细胞因子暴露的具体影响该细胞培养模型的吸引力。此外，该结果是非常可重复的和容易地实现（ 图2）。注意事项要牢记包括细胞的融合。例如，当在70〜80％，CON处理在100％汇合处理的细胞不响应，以及​​对细胞因子的相同浓度能量密度。此外，通路的细胞已经历的数量应被跟踪，因为细胞反应也可能会被影响（因为解冻超过8-12周）已经处于培养太久。而治疗的培养细胞用细胞因子的细胞活力下降，随后线粒体的健康，它决不是一个型号为败血症本身。然而，使用一个更临床相关的模型，例如使用主肾细胞从小鼠和/或治疗与刺激的THP1细胞33自然分泌的细胞因子的分离，这是工作的自然延伸。定败血症期间线粒体在肾功能衰竭的理解的间隙，例如细胞培养物的模型和评估可提供初始洞察机制，以及用于测试药物化合物的有效性作为预防性或介入治疗的初步的平台。 从培养细胞或动物组织中分离出的线粒体承受快速评估的细胞治疗或疾病进展对线粒体健康的影响的能力。的分离和评估协议不是技术上苛求来执行，并且可以很容易地进行修改，与其他的细胞系或主要组织使用。了MIB和EB用于每个样品类型是很好的起点配方作为必要的，如果一个特定的细胞或组织类型具有不同的要求，可以进行调整。例如，在加入脂肪酸游离BSA的通常是必需的与神经元的使用，因为包含在脊髓协议15,34。的细胞破碎方法也可以通过使用更多的注射器驱逐或改变手均质而不是使用一个钻头或反之亦然改变。此外，重均化所述第一离心分离沉淀的，因为完成的脊髓线粒体过程中，可能是必要的，以获得线粒体的一个令人满意的量。该隔离协议产生可行的mitochondri一个很好的指标一个是利用细胞色素C ELISA的。该测定是一个快速简便的替代细胞色素c释放的Western印迹分析，也是细胞色素c释放的治疗线粒体与肽或其它化合物19,27的后一个有用的评估。可替代地，可以TMRE协议开发过程中被使用，但以评估线粒体从已知是健康样品中分离是重要的。此外，隔离从小鼠组织线粒体未经常在文献中描述时，肝线粒体可以作为阳性对照以及用于设定一个基准。例如，在该协议中，从每一个动物，无论是在协议的开发以及实验肝脏也切除，用于线粒体隔离。用于制备另一种考虑是在分离过程结束时获得的线粒体的量。如果线粒体的浓度为1毫克/毫升或更小，然后检测结果可能被证明不可靠的（未发表意见VDGM）。无论起始原料，MIB配方或中断方法的量应移动到功能测定之前进行优化。一旦成功隔离协议达到后，线粒体也可使用在其他测定法，如总ATP含量和ATP生成13,14。 此处所描述的线粒体功能协议允许的电位传感染料TMRE线粒体吸收使用标准荧光板读数器的间接测量。后线粒体已经分离和适当的化学物质加入，它们被孵育TMRE，然后沉淀。通过处理全细胞或动物，并分离到ΔΨ评估之前线粒体，混杂变量如耐多药转运或ATP的磁通被避免。荧光的上清液中的量，然后与理解是，线粒体的健康分析更强的ΔΨ和TMRE的，因此更多的摄取。其结果是，荧光的上清液中的量应该少在健康线粒体和更多的不健康线粒体。由包括EB-仅治疗，荧光的这种控制和脱开线粒体之间的倍数差异可以计算出来。每一套反应运行另一个重要的控制是TMRE孤单。从这个控制的结果提供了最大可能的荧光读数。由于TMRE加入到反应是新鲜稀释，有内在的变化。例如，〜20,000 25,000 30,000 TMRE只读数在不同的时间协议的开发过程中实现的。如果TMRE仅信号比任何处理过的线粒体样本大得多和/或如果处理的线粒体不显著不同于未处理，则耦合线粒体的分离是最有可能不成功。在ΔΨ的降低由所获得的比率按照此协议时，类似于以前报道的数量进行控制，健康细胞2,35。的变化ΔΨ由线粒体从控制分离，未处理的HEK-293T细胞和* 3的细胞因子处理的细胞的比较表明，在MTT测定法中观察到的细胞活力下降转换为降低线粒体功能。这酶标仪检测可以很容易地与线粒体隔离从任何来源和用来检查疾病模型，如ALS使用，因为在这里13,14证明。 几项研究已经利用线粒体特异性荧光染料检测MOMP，这是在该板读数器测定的成因重要。然而，这些研究既可以使用全细胞筛选，阻止化合物或诱导MOMP 35,36，探索新的ΔΨ传感染料2或寻求其他格式，如流式细胞仪上的微芯片37。该协议是独一无二的，因为它描述的广告etailed法分离并使用通过线粒体回应称ΔΨ，改变化合物迅速调查全细胞的治疗效果。这个协议利用各种解偶联剂和抑制剂，并表明它们中的任何将适合于评估线粒体健康。因此，尽管在由于细胞应激或渐进蜂窝功能障碍的线粒体的改变这个特定调查中心，该协议也可以用于通过使用特定的化合物和/或加入，例如琥珀酸和NADH的电子源的探索改变线粒体代谢。 执行分离线粒体评估时的重要考虑因素包括治疗的卷的一致性，保持在MIB中的线粒体和稀释它们的EB的治疗会立即之前被设置，以及使用线粒体内3小时的隔离，以确保他们的ΔΨ仍维持。该选择TMRE比其他线粒体特异性荧光染料比如JC-1是由于使用小浓度和只使用一个波长分析荧光的简单的能力。可以类似地使用荧光显微镜2,38或流式细胞仪37中 ，但是在使用读板器的花费较少的时间，并且需要较少的优化35进行此方法。另外，该协议是小得多技术要求高，更快地执行，并且更容易地再现的相比，使用一个克拉克电极22（德尔Gaizo摩尔，未发表的观察结果）。滴定每个线粒体处理量可能会提供进一步的深入了解线粒体功能和浓度的优化应该执行。线粒体的反应的总体积可以调整，以适合的隔离以及必要的治疗反应的数量时所得样品的量。如果可能的话，一个标准的50μ量;升被使用;然而，小到20微升线粒体产生可靠的结果。 而ΔΨ关联到耗氧量，该测定提出不直接测量氧的消耗。而荧光氧传感器探头如MitoExpress已经开发，可能是为了避免一些使用上述一个克拉克电极引的挫折的，每个反应的价格和较大的每次测定线粒体的量的要求保持限制性的。因此，使用TMRE和古典ΔΨ解偶联剂或抑制剂的具有几个优点。再次，多个样品或多次治疗/样品，再现性，低的起始原料的要求和/或每个反应需要分离线粒体的较低量的批次分析呈现此协议的吸引力。

活细胞中的脂肪和碳水化合物的形式银行代谢能量，并使用该能量为生物合成，膜运输和移动。在胞质溶胶中通过饮食糖酵解在转换直接转化为ATP，获得一定的能量。然而，ATP产生的在细胞中的主要来源是在线粒体内通过线粒体呼吸链1利用。线粒体的体系结构提供了必要的空间取向为有效和高效的ATP的产生。线粒体具有一个双层膜由间空间，并与基体中，最里面的线粒体隔室，房屋一起的组分分离，并协调参与ATP生成的化学反应。内膜包含一系列膜结合蛋白复合物组成的呼吸链，以及ATP合酶中，蛋白质复合物带来的ADP和Pi一起用于形成ATP的。客栈ER膜被折叠成嵴和电子沿着经由细胞色素c，其中复合物之间移动间空间内的可溶电子载体呼吸链复合物通过。作为电子移动，还原当​​量发生氧化和氢离子从基质向间空间泵送。作为高离子浓度的间空间内的结果，电化学梯度建立导致跨线粒体内膜（Δψ）2的膜电位。氧气是电子传递链的最终电子受体，和氢离子从间空间流过的ATP合酶回矩阵，并在这样做的直接导致ATP的形成。这个过程的全部描述被称为氧化磷酸化。嵴的褶皱增加内层膜的表面积，从而允许内最大电子传输和ATP生产每个线粒体。的蛋白质，酶和参与氧化磷酸化的其它分子衍生自两个核和线粒体基因。线粒体含有自己的环状DNA，编码13种蛋白质，以及tRNA和必要的ATP生产3的mRNA。然而，许多更多的蛋白质是必需的，因此是核编码。大部分这些核编码蛋 ​​白质定位到线粒体基质通过在所述前体蛋白的N-末端前序列使用的，它们的进口是由Δψ4,5-驱动部分。 超出促进细胞的生物能学，线粒体也影响主要的代谢过程，例如三氯乙酸和β-氧化，通过调节钙的蜂窝信令，以及在细胞凋亡6的关键作用。具体地说，在细胞应激时，驻留在或BCL-2家族蛋白在线粒体外膜相互作用可引起线粒体外膜渗透性（MOMP）7,8。期间的MOMP，细胞色素C和其他蛋白质被释放到细胞溶胶，并连同几个胞质蛋白形成复合物称为凋亡体9,10。的凋亡体激活胱天蛋白酶是继续在细胞凋亡的执行阶段裂解细胞蛋白质和DNA。一旦发生MOMP，ΔΨ被折叠和ATP的生产暂停。因此，如凋亡启动线粒体功能受到损害，并变化ΔΨ可以关联到线粒体和细胞的健康12。而凋亡是许多疾病模型，线粒体功能和改变ΔΨ端点也可以产生有关的疾病产生和/或进展的有价值的信息。例如，线粒体的结构和功能的变化已经神经变性疾病13,14的过程中记录。 在协议中，异的第一部分完整的线粒体是保留其ΔΨ的LATION描述。 HEK-293T细胞暴露于不同浓度的重组TNF-α，IL1-β和IFN-γ的组合来诱导细胞凋亡。选择这些细胞因子，因为它们经常报道为高的原代人败血性样品15和凋亡的外源性途径中可以通过TNF-α与其受体的结合6的相互作用触发。因为有必要以比培养细胞从主组织隔离官能线粒体微妙的变化，并且由于大量的研究利用动物，该协议还描述了如何从肝脏和前侧索硬化症（ALS）的小鼠模型的脊髓分离线粒体。 该协议的第二部分被开发利用的电位敏感的荧光染料用荧光板读数器监测扰动对线粒体膜电位。差异蜂窝状态（ 即 ，健康与不健康的）之间s的通过定量与解偶联剂，呼吸链抑制剂，复杂抑制剂和离子载体，所有这些都导致线粒体膜电位的耗散一起分离线粒体的ΔΨ的强度来区分。的线粒体健康，较大处理后在ΔΨ的变化与线粒体抑制剂，因此线粒体的反应可以用作线粒体（DYS）功能的一个指标。 采用分离线粒体，而不是在原地功能评估提供了确切的证据表明，病理或治疗直接调制改变细胞器16-18。虽然有在文献中以分离从培养的细胞中线粒体的方法，它们是模糊的17和/或利用专门的设备16。该协议详细描述了隔离的方法，是易于适应其它细胞系，包括初级和组织培养13,14,19,20。许多分离的线粒体的研究利用在这个协议，但与克拉克电极中使用的相同的线粒体解偶联剂和抑制剂（ 图21是多篇论文在文献中有代表性的例子），而这又是一个非常具体的和专门的设备。此外，这种传统的方法具有如低产量和高复杂22,23限制，并且需要大量的线粒体的（〜500微克/反应）。在这个协议中，该荧光膜感应电位探针TMRE结合使用用荧光板读数器，这是一个标准的机器在许多实验室。 TMRE被广泛认为是因为它快速进入细胞并分离线粒体，可以在低浓度24中。多个反应可以迅速地设置在串联和使用此协议批分析。此外，反应s需要一个更少量孤立的线粒体（〜10微克/反应）。通过要求较少的材料，更小的组织或细胞培养物的样品可以用作一个起点线粒体隔离，更多的重复或反应可以建立起来，并有可能足够的材料为其它分离线粒体实验如ATP产生，耗氧量，或导入分析是可能的。

<table><tbody><tr><td>L-glutamic acid</td><td>Sigma</td><td>G1251</td><td>Can use the potassium salt instead.</td></tr><tr><td>malic acid</td><td>Sigma</td><td>M8304</td><td>Can use the potassium salt instead.</td></tr><tr><td>KH&lt;sub&gt;2&lt;/sub&gt;PO&lt;sub&gt;4&lt;/sub&gt;</td><td>Sigma</td><td>P0662</td><td></td></tr><tr><td>K&lt;sub&gt;2&lt;/sub&gt;HPO&lt;sub&gt;4&lt;/sub&gt;</td><td>Sigma</td><td>P3786</td><td></td></tr><tr><td>EGTA</td><td>Sigma</td><td>E3889</td><td></td></tr><tr><td>Trisma base</td><td>Sigma</td><td>T6066</td><td></td></tr><tr><td>MOPS</td><td>Sigma</td><td>M3183</td><td></td></tr><tr><td>CCCP</td><td>Sigma</td><td>C2920</td><td>Dilute down to 100&amp;nbsp;&amp;#956;M as a working stock in ethanol and store at -20&amp;nbsp;&amp;#176;C.</td></tr><tr><td>Valinomycin</td><td>Sigma</td><td>V0627</td><td>Make in DMSO and use as a 5&amp;nbsp;&amp;#956;M working stock. Store at -20&amp;nbsp;&amp;#176;C.</td></tr><tr><td>sucrose</td><td>Fisher</td><td>S5-500</td><td></td></tr><tr><td>KCN</td><td>Mallinckrodt</td><td>6379</td><td>Make a concentrated stock in ethanol and then dilute with water&amp;nbsp;</td></tr><tr><td>rotenone</td><td>Sigma</td><td>R8875</td><td>Highly toxic. Made in ethanol.</td></tr><tr><td>oligomycin</td><td>Sigma</td><td>O4876</td><td>Highly toxic. Made in ethanol.</td></tr><tr><td>ADP</td><td>Sigma</td><td>A2754</td><td></td></tr><tr><td>TMRE</td><td>Sigma</td><td>8717-25mg</td><td>Dilute 100&amp;nbsp;&amp;#956;M stock with EB immediatley before use.</td></tr><tr><td>DMEM</td><td>Gibco</td><td>11965-084</td><td>1x regular (high glucose).</td></tr><tr><td>Pen/Strep</td><td>Invitrogen</td><td>15140-155</td><td></td></tr><tr><td>L-glutamine</td><td>Fisher</td><td>SH3002101</td><td>Store aliquots at -20&amp;nbsp;&amp;#176;C</td></tr><tr><td>FBS</td><td>Lonza</td><td>14-501F</td><td>US origin, premium quality. Heat inactivate and store aliquots at -20&amp;nbsp;&amp;#176;C.</td></tr><tr><td>Trypsin-EDTA</td><td>Sigma</td><td>T4049</td><td></td></tr><tr><td>DMSO</td><td>SIgma</td><td>D2650</td><td></td></tr><tr><td>Protien Assay Dye (5x)</td><td>Bio-Rad</td><td>500-0006</td><td>Any protein assay can substitute.</td></tr><tr><td>BSA</td><td>Fisher</td><td>BP1600-100</td><td>Make 2 mg/ml stock in water for protein assay.</td></tr><tr><td>MTT powder</td><td>Sigma</td><td>M2128</td><td>Filter sterlize 5 mg/ml stock made in PBS. Store aliquots at -20&amp;nbsp;&amp;#176;C; store at 4&amp;nbsp;&amp;#176;C for up to 1 week.</td></tr><tr><td>Tergitol solution (NP-40)</td><td>Sigma</td><td>NP40S</td><td></td></tr><tr><td>Recombinant Human IL-1B</td><td>Gibco</td><td>PCH08014</td><td>Once opened store aliquots at -20&amp;nbsp;&amp;#176;C</td></tr><tr><td>Recombinant Human TNF-alpha</td><td>Gibco</td><td>PHC3015L</td><td></td></tr><tr><td>Recombinant Human IFN-gamma</td><td>Gibco</td><td>PHC4031</td><td></td></tr><tr><td>Dulbeccos PBS (-/-)</td><td>Sigma</td><td>D8537</td><td>Make sure it is without Mg&lt;sup&gt;2+&lt;/sup&gt; and Ca&lt;sup&gt;2+&lt;/sup&gt; ions.</td></tr><tr><td>Cytochrom &lt;em&gt;c&lt;/em&gt; ELISA kit</td><td>R&amp;amp;D systems</td><td>DTC0</td><td>Human for HEK-293T cells.&amp;nbsp;</td></tr></tbody></table>

isolation and functional analysis of mitochondria from cultured cells and mouse tissue

Comparison between two or more distinct groups, such as healthy vs. disease, is necessary to determine cellular status. Mitochondria are at the nexus of cell heath due to their role in both cell metabolism and energy production as well as control of apoptosis. Therefore, direct evaluation of isolated mitochondria and mitochondrial perturbation offers the ability to determine if organelle-specific (dys)function is occurring. The methods described in this protocol include isolation of intact, functional mitochondria from HEK cultured cells and mouse liver and spinal cord, but can be easily adapted for use with other cultured cells or animal tissues. Mitochondrial function assessed by TMRE and the use of common mitochondrial uncouplers and inhibitors in conjunction with a fluorescent plate reader allow this protocol not only to be versatile and accessible to most research laboratories, but also offers high throughput.

HEK-293T细胞用200微克/毫升的TNF-α，40纳克/毫升IL1-β，75纳克/毫升的IFN-γ处理（* 3）为24〜48小时，导致渐进量的细胞死亡（ 图1A ）。用MTT测定法细胞活力进行评估和一致表明，有〜10％的降低细胞生存力，24小时的治疗和〜20％的减少与48小时的治疗。具有相似的浓度（100微克/毫升的TNF-α，40纳克/毫升IL1-β，75纳克/毫升的IFN-γ）在48小时，得到类似的细胞死亡的结果，和治疗与个别的细胞因子处理的细胞显示，在减少生存能力是由于组合的影响（ 图1B）。在图1B的生存能力数据代表3个独立实验中一式三份每次运行的平均值+/-标准偏差，且误差棒的密封性演示数据的再现性。细胞因子的量可以在本治疗方法中以进一步elucidat调整E对细胞死亡的每个细胞因子的贡献，和其它细胞因子或其它因素也可包括在内，只要适当的控制被执行。 从细胞和组织中线粒体的分离已经在文献中自1940年代以来28,29说明。的前提下的程序是细胞破坏，随后通过差速离心，其中原油线粒体已知沉淀在〜10,000×克。即利用分离线粒体许多协议需要完整的双膜，可以维持它们的膜电位，因此，有必要评估完整性一旦隔离完成。在从培养的HEK细胞的线粒体隔离协议的发展，一个细胞色素C测定用于表示是否获得了完整的线粒体。使用可商购的细胞色素C的ELISA试剂盒，线粒体分离后进行量化，并稀释在EB，然后或者EB或DMSO并将该反应孵育地置于工作台顶部。然后，通过从周围的上清液中分离线粒体，两个级分的细胞色素c的内容分析被用作压力计用于线粒体膜完整性;如果大多数的细胞色素c被保持在线粒体沉淀然后线粒体被认为是完整的，而如果所述的细胞色素C的15％以上在上清液被发现，然后线粒体被认为不适合用于功能研究。如表1所示，该协议描述收率〜12％的细胞色素c释放和〜15％时，分离的线粒体是预先用DMSO。类似的结果已对线粒体从其他的细胞系或动物组织19,20,27分离以前的报告。可替代地，线粒体完整性也可以与TMRE，这是小鼠肝脏和脊髓线粒体的分离过程中使用的评估。在最初的隔离协议开发包NT，试验评估线粒体肝脏和健康的，正常小鼠线粒体脊髓孤立。对于每个样品，ΔΨ通过比较从通过EB处理，以那些孵育FCCP +缬氨霉素（ 表2）中分离的线粒体的荧光信号进行评价。 &gt; 1处理的和未处理的线粒体之间的荧光的比率作为健康的，完整的线粒体的指示。 一旦成功线粒体隔离协议被开发，线粒体从控制，未经处理的细胞中分离和* 3的细胞因子处理的细胞，或正常和SOD1小鼠进行了分析。从两组HEK线粒体进行治疗与改变TMRE吸收测量各种解偶联剂和抑制剂和ΔΨ。在控制EB折差别对待线粒体解偶联剂/抑制剂治疗的线粒体表示ΔΨ两组之间的强度差异（代表性的数据和CAL表3算结果）。此外，减少百分之从未经处理的细胞，以3 *处理过的细胞是否意味线粒体卫生用细胞因子治疗的影响，证实了细胞活力数据（ 图2）。通过使用线粒体从肝脏和四个独立的正常，对照小鼠的脊髓中分离相比4 ALS小鼠TMRE摄取评价ΔΨ强度表明该组织的健康，也可使用此协议评估。如由在ΔΨ倍差异和百分比减小，EB-治疗和FCCP +缬氨霉素处理来自对照和疾病进展的动物线粒体的比较是显著不同（ 表4;又见引用13 14）。 <img alt="图1" fo:content-width="3in" src="/files/ftp_upload/52076/52076fig1highres.jpg" width="500px" /> 图1:细胞死亡诱导D由* 3治疗是渐进随着时间的推移和大于与单独的细胞因子的治疗方法。（A）中的HEK-293T细胞处理24或48小时，细胞死亡使用MTT测定法测定。值分别标准化为对照，未处理的样品和数据代表对于n三次测量= 3，+/-标准偏差。（B）中的HEK-293T细胞用单独的细胞因子或鸡尾酒（* 3）48小时和细胞死亡评估处理通过MTT法。数据表示每组5为每个单独的细胞因子和n = 6的用于* 3，+/-标准偏差。 <table border="0" cellpadding="0" cellspacing="0"><tbody><tr><td></td><td> 颗粒（AU） </td><td> 上清液（AU） </td><td> ％细胞色素c释放 </td></tr><tr><td> 0 </td><td> 0.818 </td><td> 0.115 </td><td> 12.6±2.27 </td></tr><tr><td> DMSO </td><td> 0.793 </td><td> 0.142 </td><td> 15.3±; 2.23 </td></tr></tbody></table> 表1:细胞色素c在从培养细胞中分离线粒体保留数据表示n = 4时的平均值+/-标准偏差。 <table border="0" cellpadding="0" cellspacing="0"><tbody><tr><td></td><td></td><td> 肝1 </td><td> 肝2 </td><td> 脊髓1 </td><td> 脊髓2 </td></tr><tr><td fo:padding-top="17px" rowspan="3" style="vertical-align:middle;text-align:center"> RFU </td><td> EB </td><td> 97830 </td><td> 94132 </td><td> 339716 </td><td> 290154 </td></tr><tr><td> F / V </td><td> 435188 </td><td> 461299 </td><td> 385366 </td><td> 482480 </td></tr><tr><td> 比 </td><td> 4.45 </td><td> 4.9 </td><td> 1.13 </td><td> 1.66 </td></tr><tr><td colspan="6"> EB =实验缓冲液和F / V =​​ FCCP +缬氨霉素。 </td></tr></tbody></table> 表2:测量ΔΨ从线粒体从正常小鼠组织中分离出两个单独的动物被用于分离肝脏和脊髓线粒体串联（ 例如肝1和脊髓1顷来自同一动物）。 <table border="0" cellpadding="0" cellspacing="0"><tbody><tr><td></td><td></td><td> EB </td><td> 寡 </td><td> 腐烂 </td><td> KCN </td><td> ADP </td><td> F / V </td></tr><tr><td fo:padding-top="10px" rowspan="2" style="vertical-align:middle"> RFU </td><td> 0 </td><td> 4844 </td><td> 12507 </td><td> 12734 </td><td> 9925 </td><td> 10892 </td><td> 4844 </td></tr><tr><td>* 3 </td><td> 6517 </td><td> 13,639 </td><td> 12435 </td><td> 10235 </td><td> 13154 </td><td> 6517 </td></tr><tr><td fo:padding-top="9px" rowspan="2" style="vertical-align:middle"> 倍的差异 </td><td> 0 </td><td> NA </td><td> 2.58 </td><td> 2.63 </td><td> 2.05 </td><td> 2.25 </td><td> 2.65 </td></tr><tr><td> * 3 </td><td> NA </td><td> 2.09 </td><td> 1.91 </td><td> 1.57 </td><td> 2.02 </td><td> 2.18 </td></tr><tr><td> ％跌幅 </td><td> 0与* 3 </td><td></td><td> 18.94 </td><td> 27.41 </td><td> 23.35 </td><td> 10.24 </td><td> 17.67 </td></tr><tr><td colspan="8"> EB =实验缓冲;寡寡=;腐鱼藤酮=; KCN =氰化钾和F / V =​​ FCCP +霉素。 </td></tr></tbody></table> 表3:原始数据和计算从小区Culture孤立的线粒体实验。 <img alt="图2" fo:content-width="3in" src="/files/ftp_upload/52076/52076fig2highres.jpg" width="500px" /> 图2:线粒体从细胞因子处理的细胞中分离出已下降的膜电位 。减少百分之ΔΨ的为0与* 3对每个治疗与对照组相比，作为计算在表2中 。数据表示对于n重复反应的平均值= 3（寡霉素，鱼藤酮，和FCCP /缬氨霉素）或n = 2（ KCN和ADP）+/-标准偏差。基于倍的差距计算，* =统计显著的p值; p值= 0.03，0.01，和0.05寡，鱼藤酮和FCCP /缬氨霉素，分别。 <table border="0" cellpadding="0" cellspacing="0"><tbody><tr><td></td><td></td><td> 脊髓 </td><td> 肝 </td></tr><tr><td fo:padding-top="11像素"ROWSPAN ="2"的风格="垂直对齐:中间"&gt; 倍的差异 </td><td> 控制 </td><td> 1.54±0.48 </td><td> 3.20±1.56 </td></tr><tr><td> SOD1 </td><td> 1.10±0.30 </td><td> 3.12±1.75 </td></tr><tr><td> ％跌幅 </td><td></td><td> 28.1 </td><td> 2.48 </td></tr><tr><td> p值 </td><td></td><td> 0.03 </td><td> 0.41 </td></tr></tbody></table> 表4:从线粒体的SOD1小鼠的脊髓中分离出已经作为评估FCCP +缬氨霉素数据表示每组4只动物的平均膜电位降低 。

Watch this Scientific Journal Video about Isolation and Functional Analysis of Mitochondria from Cultured Cells and Mouse Tissue at JoVE.com

Isolation and Functional Analysis of Mitochondria from Cultured Cells and Mouse Tissue

Comparison of mitochondrial membrane potential between samples yields valuable information about cellular status. Detailed steps for isolating mitochondria and assessing response to inhibitors and uncouplers using fluorescence are described. The method and utility of this protocol are illustrated by use of a cell culture and animal model of cellular stress.

隔离和线粒体从培养细胞和小鼠组织功能分析

Comparison between two or more distinct groups, such as healthy vs. disease, is necessary to determine cellular status. Mitochondria are at the nexus of ...

isolation-functional-analysis-mitochondria-from-cultured-cells-mouse

Universidad San Sebastian

Research

JoVE Journal

Biology

39.3K 次观看.  Elon University. Comparison of mitochondrial membrane potential between samples yields valuable information about cellular status. Detailed steps for isolating mitochondria and assessing response to inhibitors and uncouplers using fluorescence are described. The method and utility of this protocol are illustrated by use of a cell culture and animal model of cellular stress. 

视频: Isolation and Functional Analysis of Mitochondria from Cultured Cells and Mouse Tissue

Isolation and Analysis of Hematopoietic Stem Cells from the Placenta

Hematopoietic stem cells (HSCs) have the ability to self-renew and generate all cell types of the blood lineages throughout the lifetime of an individual. All HSCs emerge during embryonic development, after which their pool size is maintained by self-renewing cell divisions. Identifying the anatomical origin of HSCs and the critical developmental events regulating the process of HSC development has been complicated as many anatomical sites participate during fetal hematopoiesis. Recently, we identified the placenta as a major hematopoietic organ where HSCs are generated and expanded in unique microenvironmental niches (Gekas, et al 2005, Rhodes, et al 2008). Consequently, the placenta is an important source of HSCs during their emergence and initial expansion.
In this article, we show dissection techniques for the isolation of murine placenta from E10.5 and E12.5 embryos, corresponding to the developmental stages of initiation of HSCs and the peak in the size of the HSC pool in the placenta, respectively. In addition, we present an optimized protocol for enzymatic and mechanical dissociation of placental tissue into single-cell suspension for use in flow cytometry or functional assays. We have found that use of collagenase for single-cell suspension of placenta gives sufficient yields of HSCs. An important factor affecting HSC yield from the placenta is the degree of mechanical dissociation prior to, and duration of, enzymatic treatment.
We also provide a protocol for the preparation of fixed-frozen placental tissue sections for the visualization of developing HSCs by immunohistochemistry in their precise cellular niches. As hematopoietic specific antigens are not preserved during preparation of paraffin embedded sections, we routinely use fixed frozen sections for localizing placental HSCs and progenitors.

We have identified the placenta as a major hematopoietic organ during development. We found that hematopoietic stem cells (HSCs) are both generated and expanded in the placenta in unique microenvironmental niches. Here, we describe experimental techniques required for isolation and visualization of HSCs in the mouse placenta.

从胎盘中的造血干细胞的分离和分析

Hematopoietic stem cells (HSCs) have the ability to self-renew and generate all cell types of the blood lineages throughout the lifetime of an individual. ...

科研

生物学

Purification of Mitochondria from Yeast Cells

Mitochondria are the main site of ATP production during aerobic metabolism in eukaryotic non-photosynthetic cells1. These complex organelles also play essential roles in apoptotic cell death2, cell survival3, mammalian development4, neuronal development and function4, intracellular signalling5, and longevity regulation6. Our understanding of these complex biological processes controlled by mitochondria relies on robust methods for assessing their morphology, their protein and lipid composition, the integrity of their DNA, and their numerous vital functions. The budding yeast Saccharomyces cerevisiae, a genetically and biochemically manipulable unicellular eukaryote with annotated genome and well-defined proteome, is a valuable model for studying the molecular and cellular mechanisms underlying essential biological functions of mitochondria. For these types of studies, it is crucial to have highly pure mitochondria. Here we present a detailed description of a rapid and effective method for purification of yeast mitochondria. This method enables the isolation of highly pure mitochondria that are essentially free of contamination by other organelles and retain their structural and functional integrity after their purification. Mitochondria purified by this method are suitable for cell-free reconstitution of essential mitochondrial processes and can be used for the analysis of mitochondrial structure and functions, mitochondrial proteome and lipidome, and mitochondrial DNA.

We describe a rapid and effective method for purification of mitochondria from the yeast Saccharomyces cerevisiae. This method enables the high-yield isolation of pure mitochondria that are essentially free of contamination by other organelles and retain their structural and functional integrity after their purification.

从酵母细胞线粒体的纯化

Mitochondria are the main site of ATP production during aerobic metabolism in eukaryotic non-photosynthetic cells1. These complex organelles also play ...

Mitochondrial Isolation from Skeletal Muscle

Mitochondria are organelles controlling the life and death of the cell. They participate in key metabolic reactions, synthesize most of the ATP, and regulate a number of signaling cascades2,3. Past and current researchers have isolated mitochondria from rat and mice tissues such as liver, brain and heart4,5. In recent years, many researchers have focused on studying mitochondrial function from skeletal muscles.
Here, we describe a method that we have used successfully for the isolation of mitochondria from skeletal muscles 6. Our procedure requires that all buffers and reagents are made fresh and need about 250-500 mg of skeletal muscle. We studied mitochondria isolated from rat and mouse gastrocnemius and diaphragm, and rat extraocular muscles. Mitochondrial protein concentration is measured with the Bradford assay. It is important that mitochondrial samples be kept ice-cold during preparation and that functional studies be performed within a relatively short time (~1 hr). Mitochondrial respiration is measured using polarography with a Clark-type electrode (Oxygraph system) at 37&deg;C7. Calibration of the oxygen electrode is a key step in this protocol and it must be performed daily. Isolated mitochondria (150 &mu;g) are added to 0.5 ml of experimental buffer (EB). State 2 respiration starts with addition of glutamate (5mM) and malate (2.5 mM). Then, adenosine diphosphate (ADP) (150 &mu;M) is added to start state 3. Oligomycin (1 &mu;M), an ATPase synthase blocker, is used to estimate state 4. Lastly, carbonyl cyanide p-[trifluoromethoxy]-phenyl-hydrazone (FCCP, 0.2 &mu;M) is added to measurestate 5, or uncoupled respiration 6. The respiratory control ratio (RCR), the ratio of state 3 to state 4, is calculated after each experiment. An RCR &ge;4 is considered as evidence of a viable mitochondria preparation.
In summary, we present a method for the isolation of viable mitochondria from skeletal muscles that can be used in biochemical (e.g., enzyme activity, immunodetection, proteomics) and functional studies (mitochondrial respiration).

This protocol describes a procedure to study the respiration of mitochondria isolated from skeletal muscles. This method was adapted from Scorrano et al. (2007). The mitochondrial isolation procedure requires about 2 hours. The mitochondrial respiration can be completed in about 1 hour.

骨骼肌的线粒体分离

Mitochondria are organelles controlling the life and death of the cell. They participate in key metabolic reactions, synthesize most of the ATP, and ...

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 ...

生物化学

<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 ...

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

Previously described mitochondrial isolation methods using differential centrifugation and/or Ficoll gradient centrifugation require 60 to 100 min to complete. We describe a method for the rapid isolation of mitochondria from mammalian biopsies using a commercial tissue dissociator and differential filtration. In this protocol, manual homogenization is replaced with the tissue dissociator&#8217;s standardized homogenization cycle. This allows for uniform and consistent homogenization of tissue that is not easily achieved with manual homogenization. Following tissue dissociation, the homogenate is filtered through nylon mesh filters, which eliminate repetitive centrifugation steps. As a result, mitochondrial isolation can be performed in less than 30 min. This isolation protocol yields approximately 2 x 1010 viable and respiration competent mitochondria from 0.18 &#177; 0.04 g (wet weight) tissue sample.

A method for rapid isolation of mitochondria from mammalian tissue biopsies is described. Rat liver or skeletal muscle preparations were homogenized with a commercial tissue dissociator and mitochondria were isolated by differential filtration through nylon mesh filters. Mitochondrial isolation time is &#60;30 min compared to 60 - 100 min using alternative methods.

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 ...

隔离和线粒体从培养细胞和小鼠组织功能分析

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此视频中的章节

从胎盘中的造血干细胞的分离和分析

从酵母细胞线粒体的纯化

骨骼肌的线粒体分离

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

从啮齿动物心脏组织中稳健地分离线粒体

从啮齿动物心脏组织中稳健地分离线粒体

快速分离纯化线粒体移植用组织分离和鉴别过滤