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?...

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 border="0" cellpadding="0" cellspacing="0" width="686">
	<tbody>
		<tr height="42">
			<td height="42" style="height:42px;width:209px;">Name of Material/ Equipment</td>
			<td style="width:137px;">Company</td>
			<td style="width:163px;">Catalog Number</td>
			<td style="width:179px;">Comments/Description</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">L-glutamic acid</td>
			<td>Sigma</td>
			<td>G1251</td>
			<td>Can use the potassium salt instead.</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">malic acid</td>
			<td>Sigma</td>
			<td>M8304</td>
			<td>Can use the potassium salt instead.</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">KH2PO4</td>
			<td>Sigma</td>
			<td>P0662</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">K2HPO4</td>
			<td>Sigma</td>
			<td>P3786</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">EGTA</td>
			<td>Sigma</td>
			<td>E3889</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Trisma base</td>
			<td>Sigma</td>
			<td>T6066</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">MOPS</td>
			<td>Sigma</td>
			<td>M3183</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">CCCP</td>
			<td>Sigma</td>
			<td>C2920</td>
			<td>Dilute down to 100uM as a working stock in ethanol and store at -20C.&#160;</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Valinomycin</td>
			<td>Sigma</td>
			<td>V0627</td>
			<td>Make in DMSO and use as a 5uM working stock. Store at -20C.</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">sucrose</td>
			<td>Fisher</td>
			<td>S5-500</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">KCN</td>
			<td>Mallinckrodt</td>
			<td align="right">6379</td>
			<td>Make a concentrated stock in ethanol and then dilute with water&#160;</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">rotenone</td>
			<td>Sigma</td>
			<td>R8875</td>
			<td>Highly toxic. Made in ethanol.</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">oligomycin</td>
			<td>Sigma</td>
			<td>O4876</td>
			<td>Highly toxic. Made in ethanol.</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">ADP</td>
			<td>Sigma</td>
			<td>A2754</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">TMRE</td>
			<td>Sigma</td>
			<td>8717-25mg</td>
			<td>Dilute 100uM stock with EB immediatley before use.</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">DMEM</td>
			<td>Gibco</td>
			<td>11965-084</td>
			<td>1x regular (high glucose).</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Pen/Strep</td>
			<td>Invitrogen</td>
			<td>15140-155</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">L-glutamine</td>
			<td>Fisher</td>
			<td>SH3002101</td>
			<td>Store aliquots at -20C</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">FBS</td>
			<td>Lonza</td>
			<td>14-501F</td>
			<td>US origin, premium quality. Heat inactivate and store aliquots at -20</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Trypsin-EDTA</td>
			<td>Sigma</td>
			<td>T4049</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">DMSO</td>
			<td>SIgma</td>
			<td>D2650</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:209px;">Protien Assay Dye (5x)</td>
			<td style="width:137px;">Bio-Rad</td>
			<td style="width:163px;">500-0006</td>
			<td>Any protein assay can substitute.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:209px;">BSA</td>
			<td style="width:137px;">Fisher</td>
			<td style="width:163px;">BP1600-100</td>
			<td>Make 2mg/mL stock in water for protein assay.</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">MTT powder</td>
			<td>Sigma</td>
			<td>M2128</td>
			<td>Filter sterlize 5mg/mL stock made in PBS. Store aliquots at -20C; store at 4C for up to 1 week.</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Tergitol solution (NP-40)</td>
			<td>Sigma</td>
			<td>NP40S</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Recombinant Human IL-1B</td>
			<td>Gibco</td>
			<td>PCH08014</td>
			<td>Once opened store aliquots at -20C</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Recombinant Human TNF-alpha</td>
			<td>Gibco</td>
			<td>PHC3015L</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Recombinant Human IFN-gamma</td>
			<td style="width:137px;">Gibco</td>
			<td>PHC4031</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:209px;">Dulbeccos PBS (-/-)</td>
			<td>Sigma</td>
			<td style="width:163px;">D8537</td>
			<td>Make sure it is without Mg2+ and Ca2+ ions.</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Cytochrom c ELISA kit</td>
			<td>R&#38;D systems</td>
			<td>DTC0</td>
			<td>Human for HEK-293T cells.&#160;</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...

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

Research

JoVE Journal

Biology

39.2K 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 ...

Dissection of Hippocampal Dentate Gyrus from Adult Mouse

The hippocampus is one of the most widely studied areas in the brain because of its important functional role in memory processing and learning, its remarkable neuronal cell plasticity, and its involvement in epilepsy, neurodegenerative diseases, and psychiatric disorders. The hippocampus is composed of distinct regions; the dentate gyrus, which comprises mainly granule neurons, and Ammon's horn, which comprises mainly pyramidal neurons, and the two regions are connected by both anatomic and functional circuits. Many different mRNAs and proteins are selectively expressed in the dentate gyrus, and the dentate gyrus is a site of adult neurogenesis; that is, new neurons are continually generated in the adult dentate gyrus. To investigate mRNA and protein expression specific to the dentate gyrus, laser capture microdissection is often used. This method has some limitations, however, such as the need for special apparatuses and complicated handling procedures. In this video-recorded protocol, we demonstrate a dissection technique for removing the dentate gyrus from adult mouse under a stereomicroscope. Dentate gyrus samples prepared using this technique are suitable for any assay, including transcriptomic, proteomic, and cell biology analyses. We confirmed that the dissected tissue is dentate gyrus by conducting real-time PCR of dentate gyrus-specific genes, tryptophan 2,3-dioxygenase (TDO2) and desmoplakin (Dsp), and Ammon's horn enriched genes, Meis-related gene 1b (Mrg1b) and TYRO3 protein tyrosine kinase 3 (Tyro3). The mRNA expressions of TDO2 and Dsp in the dentate gyrus samples were detected at obviously higher levels, whereas Mrg1b and Tyro3 were lower levels, than those in the Ammon's horn samples. To demonstrate the advantage of this method, we performed DNA microarray analysis using samples of whole hippocampus and dentate gyrus. The mRNA expression of TDO2 and Dsp, which are expressed selectively in the dentate gyrus, in the whole hippocampus of alpha-CaMKII+/- mice, exhibited 0.037 and 0.10-fold changes compared to that of wild-type mice, respectively. In the isolated dentate gyrus, however, these expressions exhibited 0.011 and 0.021-fold changes compared to that of wild-type mice, demonstrating that gene expression changes in dentate gyrus can be detected with greater sensitivity. Taken together, this convenient and accurate dissection technique can be reliably used for studies focused on the dentate gyrus.

A dissection technique for removal of the dentate gyrus from adult mouse under a stereomicroscope was demonstrated in this video-recorded protocol.

从成年小鼠海马齿状回的剖析

The hippocampus is one of the most widely studied areas in the brain because of its important functional role in memory processing and learning, its ...

Isolation of Primary Myofibroblasts from Mouse and Human Colon Tissue

The myofibroblast is a stromal cell of the gastrointestinal (GI) tract that has been gaining considerable attention for its critical role in many GI functions. While several myofibroblast cell lines are commercially available to study these cells in vitro, research results from a cell line exposed to experimental cell culture conditions have inherent limitations due to the overly reductionist nature of the work. Use of primary myofibroblasts offers a great advantage in terms of confirming experimental findings identified in a cell line. Isolation of primary myofibroblasts from an animal model allows for the study of myofibroblasts under conditions that more closely mimic the disease state being studied. Isolation of primary myofibroblasts from human colon tissue provides arguably the most relevant experimental data, since the cells come directly from patients with the underlying disease. We describe a well-established technique that can be utilized to isolate primary myofibroblasts from both mouse and human colon tissue. These isolated cells have been characterized to be alpha-smooth muscle actin and vimentin-positive, and desmin-negative, consistent with subepithelial intestinal myofibroblasts. Primary myofibroblast cells can be grown in cell culture and used for experimental purposes over a limited number of passages.

The myofibroblast is an influential stromal cell of the gastrointestinal tract that regulates important physiologic processes in both normal and disease states. We describe a technique that allows for the isolation of primary myofibroblasts from both mouse and human colon tissue, which can be utilized for in vitro experimentation.

从小鼠和人类结肠组织初级肌成纤维细胞的分离

The myofibroblast is  a stromal cell of the gastrointestinal (GI) tract that has been gaining  considerable attention for its critical role in many GI ...

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 ...

Isolation of Mitochondria from Minimal Quantities of Mouse Skeletal Muscle for High Throughput Microplate Respiratory Measurements

Dysfunctional skeletal muscle mitochondria play a role in altered metabolism observed with aging, obesity and Type II diabetes. Mitochondrial respirometric assays from isolated mitochondrial preparations allow for the assessment of mitochondrial function, as well as determination of the mechanism(s) of action of drugs and proteins that modulate metabolism. Current isolation procedures often require large quantities of tissue to yield high quality mitochondria necessary for respirometric assays. The methods presented herein describe how high quality purified mitochondria (~ 450 &#181;g) can be isolated from minimal quantities (~75-100 mg) of mouse skeletal muscle for use in high throughput respiratory measurements. We determined that our isolation method yields 92.5&#177; 2.0% intact mitochondria by measuring citrate synthase activity spectrophotometrically. In addition, Western blot analysis in isolated mitochondria resulted in the faint expression of the cytosolic protein, GAPDH, and the robust expression of the mitochondrial protein, COXIV. The absence of a prominent GAPDH band in the isolated mitochondria is indicative of little contamination from non-mitochondrial sources during the isolation procedure. Most importantly, the measurement of O2 consumption rate with micro-plate based technology and determining the respiratory control ratio (RCR) for coupled respirometric assays shows highly coupled (RCR; &#62;6 for all assays) and functional mitochondria. In conclusion, the addition of a separate mincing step and significantly reducing motor driven homogenization speed of a previously reported method has allowed the isolation of high quality and purified mitochondria from smaller quantities of mouse skeletal muscle that results in highly coupled mitochondria that respire with high function during microplate based respirometirc assays.

Here, we present a modification of a previously reported method that allows for the isolation of high quality and purified mitochondria from smaller quantities of mouse skeletal muscle. This procedure results in highly coupled mitochondria that respire with high function during microplate based respirometirc assays.

从小鼠骨骼肌高通量微孔板呼吸测量最低数量的隔离线粒体

Dysfunctional skeletal muscle mitochondria play a role in altered metabolism observed with aging, obesity and Type II diabetes. Mitochondrial ...

Isolation of High Quality Murine Atrial and Ventricular Myocytes for Simultaneous Measurements of Ca2+ Transients and L-Type Calcium Current

Mouse models play a crucial role in arrhythmia research and allow studying key mechanisms of arrhythmogenesis including altered ion channel function and calcium handling. For this purpose, atrial or ventricular cardiomyocytes of high quality are necessary to perform patch-clamp measurements or to explore calcium handling abnormalities. However, the limited yield of high-quality cardiomyocytes obtained by current isolation protocols does not allow both measurements in the same mouse. This article describes a method to isolate high-quality murine atrial and ventricular myocytes via retrograde enzyme-based Langendorff perfusion, for subsequent simultaneous measurements of calcium transients and L-type calcium current from one animal. Mouse hearts are obtained, and the aorta is rapidly cannulated to remove blood. Hearts are then initially perfused with a calcium-free solution (37 &#176;C) to dissociate the tissue at the level of intercalated discs and afterwards with an enzyme solution containing little calcium to disrupt extracellular matrix (37 &#176;C). The digested heart is subsequently dissected into atria and ventricles. Tissue samples are chopped into small pieces and dissolved by carefully pipetting up and down. The enzymatic digestion is stopped, and cells are stepwise reintroduced to physiologic calcium concentrations. After loading with a fluorescent Ca2+-indicator, isolated cardiomyocytes are prepared for simultaneous measurement of calcium currents and transients. Additionally, isolation pitfalls are discussed and patch-clamp protocols and representative traces of L-type calcium currents with simultaneous calcium transient measurements in atrial and ventricular murine myocytes isolated as described above are provided.

Isolation of High Quality Murine Atrial and Ventricular Myocytes for Simultaneous Measurements of Ca2+ Transients and L-Type Calcium Current

Mouse models allow studying key mechanisms of arrhythmogenesis. For this purpose, high quality cardiomyocytes are necessary to perform patch-clamp measurements. Here, a method to isolate murine atrial and ventricular myocytes via retrograde enzyme-based Langendorff perfusion, which allows simultaneous measurements of calcium-transients and L-type calcium current, is described.

分离高质量鼠心房和心室肌细胞，同时测量 Ca2+ 瞬变和 L 型钙电流

Mouse models play a crucial role in arrhythmia research and allow studying key mechanisms of arrhythmogenesis including altered ion channel function and ...

Isolation of Mitochondria from Mouse Skeletal Muscle for Respirometric Assays

Most of the cell&#39;s energy is obtained through the degradation of glucose, fatty acids, and amino acids by different pathways that converge on the mitochondrial oxidative phosphorylation (OXPHOS) system, which is regulated in response to cellular demands. The lipid molecule Coenzyme Q (CoQ) is essential in this process by transferring electrons to complex III in the electron transport chain (ETC) through constant oxidation/reduction cycles. Mitochondria status and, ultimately, cellular health can be assessed by measuring ETC oxygen consumption using respirometric assays. These studies are typically performed in established or primary cell lines that have been cultured for several days. In both cases, the respiration parameters obtained may have deviated from normal physiological conditions in&#160;any given organ or tissue.
Additionally, the intrinsic characteristics of cultured single fibers isolated from skeletal muscle impede this type of analysis. This paper presents an updated and detailed protocol for the analysis of respiration in freshly isolated mitochondria from mouse skeletal muscle. We also provide solutions to potential problems that could arise at any step of the process. The method presented here could be applied to compare oxygen consumption rates in diverse transgenic mouse models and study the mitochondrial response to drug treatments or other factors such as aging or sex. This is a feasible method to respond to crucial questions about mitochondrial bioenergetics metabolism and regulation.

Here, we describe a detailed method for mitochondria isolation from mouse skeletal muscle and the subsequent analysis of respiration by Oxygen Consumption Rate (OCR) using microplate-based respirometric assays. This pipeline can be applied to study the effects of multiple environmental or genetic interventions on mitochondrial metabolism.

从小鼠骨骼肌中分离线粒体用于呼吸测定

Most of the cell&#39;s energy is obtained through the degradation of glucose, fatty acids, and amino acids by different pathways that converge on the ...

Isolation and Functional Analysis of Arteriolar Endothelium of Mouse Brain Parenchyma

Cerebral blood flow is conveyed by vascular resistance arteries and downstream parenchymal arterioles. Steady-state vascular resistance to blood flow increases with decreasing diameter from arteries to arterioles that ultimately feed into capillaries. Due to their smaller size and location in the parenchyma, arterioles have been relatively understudied and with less reproducibility in findings than surface pial arteries. Regardless, arteriolar endothelial cell structure and function&#8212;integral to the physiology and etiology of chronic degenerative diseases&#8212;requires extensive investigation. In particular, emerging evidence demonstrates that compromised endothelial function precedes and exacerbates cognitive impairment and dementia.
In the parenchymal microcirculation, endothelial K+ channel function is the most robust stimulus to finely control the spread of vasodilation to promote increases in blood flow to areas of neuronal activity. This paper illustrates a refined method for freshly isolating intact and electrically coupled endothelial &#34;tubes&#34; (diameter, ~25 &#181;m) from mouse brain parenchymal arterioles. Arteriolar endothelial tubes are secured during physiological conditions (37 &#176;C, pH 7.4) to resolve experimental variables that encompass K+ channel function and their regulation, including intracellular Ca2+ dynamics, changes in membrane potential, and membrane lipid regulation. A distinct technical advantage versus arterial endothelium is the enhanced morphological resolution of cell and organelle (e.g., mitochondria) dimensions, which expands the usefulness of this technique. Healthy cerebral perfusion throughout life entails robust endothelial function in parenchymal arterioles, directly linking blood flow to the fueling of neuronal and glial activity throughout precise anatomical regions of the brain. Thus, it is expected that this method will significantly advance the general knowledge of vascular physiology and neuroscience concerning the healthy and diseased brain.

Intensive preparation of intact mouse cerebral endothelial &#34;tubes&#34; from cerebral parenchymal arterioles is illustrated for studying cerebral blood flow regulation. Further, we demonstrate the experimental strengths of this endothelial study model for fluorescence imaging and electrophysiology measurement of key cellular signaling pathways, including changes in intracellular [Ca2+] and membrane potential.

小鼠脑实质小动脉内皮的分离及功能分析

Cerebral blood flow is conveyed by vascular resistance arteries and downstream parenchymal arterioles. Steady-state vascular resistance to blood flow ...

Isolation of Conditionally Immortalized Mouse Glomerular Endothelial Cells with Fluorescent Mitochondria

Glomerular endothelial cell (GEC) dysfunction can initiate and contribute to glomerular filtration barrier breakdown. Increased mitochondrial oxidative stress has been suggested as a mechanism resulting in GEC dysfunction in the pathogenesis of some glomerular diseases. Historically the isolation of GECs from in vivo models has been notoriously challenging due to difficulties in isolating pure cultures from glomeruli. GECs have complex growth requirements in vitro and a very limited lifespan. Here, we describe the procedure for isolating and culturing conditionally immortalized GECs with fluorescent mitochondria, enabling the tracking of mitochondrial fission and fusion events. GECs were isolated from the kidneys of a double transgenic mouse expressing the thermolabile SV40 TAg (from the Immortomouse), conditionally promoting proliferation and suppressing cell differentiation, and a photo-convertible fluorescent protein (Dendra2) in all mitochondria (from the photo-activatable mitochondria [PhAMexcised] mouse). The stable cell line generated allows for cell differentiation after inactivation of the immortalizing SV40 TAg gene and photo-activation of a subset of mitochondria causing a switch in fluorescence from green to red. The use of mitoDendra2-GECs allows for live imaging of fluorescent mitochondria&#39;s distribution, fusion, and fission events without staining the cells.

The article describes the method for isolating conditionally immortalized glomerular endothelial cells from the kidneys of transgenic mice expressing the thermolabile simian virus 40 and photo-activatable mitochondria, PhAMexcised. We describe the procedure for glomeruli isolation from whole kidneys using beads, digestion steps, seeding, and culturing of GECs-CD31 positive.

用荧光线粒体分离条件永生化的小鼠肾小球内皮细胞

Glomerular endothelial cell (GEC) dysfunction can initiate and contribute to glomerular filtration barrier breakdown. Increased mitochondrial oxidative ...