Name: high-resolution respirometry to assess mitochondrial function in permeabilized and intact cells
Uploaded: 2017-02-08T13:00:00.000+00:00
Duration: 8 min 33 s
Description: Watch this Scientific Journal Video about High-resolution Respirometry to Assess Mitochondrial Function in Permeabilized and Intact Cells at JoVE.com

This study was supported by the Swiss National Science Foundation (Grant n&#186; 32003B_127619).

1.细胞培养<ol><li>培养人肝癌HepG2细胞6 25厘米的Dulbecco含有10％热灭活的胎牛血清（FBS）和1％青霉素-链霉素，在37℃的恒温箱中（5％CO改良的Eagle培养基（DMEM）2细胞培养烧瓶2，95％空气）（接种密度:1×10每25 6个细胞cm 2的细胞培养瓶中，在37℃培养箱培养时间:48小时，在汇合的细胞密度:每25厘米4-5×10 6细胞2细胞培养瓶）。 </li><li>当细胞是90％至95％汇合进行实验。 </li></ol>极谱氧传感器2.高分辨率呼​​吸测量校准<ol><li>吸管2.1毫升呼吸缓冲成oxygraph室中，并在37℃下连续地使用存在于所述腔室（700转）磁性搅拌棒1小时，直到稳定的氧通量信号搅拌该缓冲得到极谱氧传感器。 注:每个oxygraph室测量氧气浓度范围内极谱氧电极和各室中计算耗氧量（光通量）。氧浓度和耗氧速率（流量）正在使用的数据采集和分析软件显示实时在线在计算机中。 </li><li>根据制造商的协议14执行极谱氧传感器的空气校准。 注意:仅每天一次在早晨进行在空气饱和实验温度在呼吸介质和氧气浓度极谱氧传感器在介质的校准。事后媒体可从腔室并重新悬浮于新鲜的呼吸媒体细胞去除被加入到oxygraph室和呼吸速率进行测量。第一个实验后，腔室可被洗涤并可以在叔进行额外系列的实验他同室而无需进一步校准。 </li></ol> 3.胰蛋白酶消化贴壁细胞，细胞计数的<ol><li>在实验的当天，吸距离为25cm 2细胞培养瓶中的DMEM中。 </li><li>冲洗在培养烧瓶中的细胞培养物单层用5ml磷酸盐缓冲盐水（PBS）中。 </li><li>吸管加入0.5ml 25mg / ml的胰蛋白酶溶液（在37℃水浴中预热至37℃）到培养烧瓶中的细胞单层，并在培养箱（5％CO 2孵育5分钟，在37℃ ，95％空气）。 </li><li>吸管5ml含DMEM 10％胎牛血清成在细胞培养烧瓶中的分离的细胞，并通过移液悬浮细胞。 </li><li>转移在室温下以350 xg离心悬浮细胞至15ml离心管中并离心5分钟，倒出上清液。 </li><li>重悬细胞沉淀在1毫升呼吸缓冲13（T 能够1）的。 </li><l<html> I&gt;使用细胞计数器计数细胞，并在呼吸缓冲器重悬至1×10 6个细胞/ ml的终密度。由于细胞呼吸速率将被标准化为细胞数，计数一个准确和精确的细胞计数细胞。 </li></ol> 4.高分辨率呼​​吸测量<ol><li>空气校准后（每日只执行一次，步骤2.1-2.2）中，从oxygraph的腔室吸出呼吸介质和来自步骤3.7至腔室加2.1的细胞悬浮液（1×10 6个细胞/ ml）的溶液中。 </li><li>由止动器的插入关闭oxygraph室中。 </li><li>搅拌该细胞在37℃下连续地使用存在于所述腔室（700转）磁性搅拌棒和直到一个稳定的氧通量信号实现记录细胞呼吸5-10分钟。 注意:氧浓度和氧通量信号正在使用的数据采集和分析软件显示实时在线在计算机S ="外部参照"&gt; 14。 </li><li>之后，注入底物和抑制剂用于通过使用以下协议的挡块的钛喷射口线粒体呼吸。 </li></ol> 5.滴定毛地黄皂苷通过呼吸测量完整细胞<ol><li>以下步骤4.1到协议4.3中描述的方法制备的含oxygraph腔细胞悬浮液（1×10 6 / ml）中。 </li><li>用注射器注入2微升0.2mM的鱼藤酮（0.2μM）"警告"的成通过室止动件的钛注射口含细胞悬液oxygraph腔和直到一个稳定的氧通量信号实现记录细胞呼吸5-10分钟。 注意:在下面的步骤的所有注射通过使用注射器塞子的钛喷射口进行。鱼藤酮的加成是可选的（它可以防止反向电子流），并且可以对毛地黄皂苷滴定实验中省略，参见步骤6.3。 </li><li>注射20微升的1M琥珀酸酯（10毫摩尔）进oxygraph腔和直到一个稳定的氧通量信号实现记录细胞呼吸5-10分钟。 </li><li>注入10微升的0.5M ADP（2.5毫摩尔）到oxygraph腔和直到一个稳定的氧通量信号实现记录细胞呼吸5-10分钟。 </li><li>注射2微升2毫米洋地黄（2微米）到oxygraph室，直到一个稳定的氧信号实现了创记录的细胞呼吸2-5分钟。 </li><li>再次注入2微升2毫米的毛地黄皂苷进入oxygraph室，直到一个稳定的氧信号实现了创记录的细胞呼吸2-5分钟。 注:逐步加成毛地黄皂苷的向细胞会诱发在蜂窝氧消耗的增加和氧通量信号将增加。 </li><li>继续注射2-4微升2mM的毛地黄皂苷逐步（2-4微米的每一步）到腔室。每个步骤之后，记录细胞呼吸2-5米在直到一个稳定的氧通量信号得以实现。 注:停止注入毛地黄皂苷当氧通量信号达到最大水平和毛地黄皂苷的进一步注射不增加呼吸率。读者应该使用自己的试剂和使用得到最佳洋地黄浓度的步骤6.2和以下协议7.2他们的实验确定在他们的实验室最佳洋地黄浓度。 </li></ol> 6.评估线粒体外膜完整性:细胞色素C <ol><li>以下步骤4.1到协议4.3中描述的方法制备的含oxygraph腔细胞悬浮液（1×10 6 / ml）中。 </li><li>注入2微升8毫毛地黄皂苷（8微米）到含有细胞悬浮液（1×10 6 /毫升）的oxygraph腔和透化细胞5分钟。 </li><li>注射20微升1M的琥珀酸酯（10毫米）进入oxygraph室和记录细胞respirat直到一个稳定的氧通量信号实现离子5-10分钟。 </li><li>注入10微升的0.5M ADP（2.5毫摩尔）到oxygraph腔和直到一个稳定的氧通量信号实现记录细胞呼吸5-10分钟。 注意:ADP的添加到细胞中会刺激复合I和诱导耗氧量的增加，并且氧通量信号将增加并稳定下来。 </li><li>注入5μl的4mM的色素c（10μM）的进oxygraph腔和直到一个稳定的氧通量信号实现记录细胞呼吸5-10分钟。 </li><li>最后注入1微升4毫克/毫升寡（2微克/毫升）和直到一个稳定的氧通量信号实现记录细胞呼吸。 </li></ol> 7.最大ADP刺激呼吸透HepG2细胞（国3） <ol><li>以下步骤4.1到协议4.3中描述的方法制备的含oxygraph腔细胞悬浮液（1×10 6 / ml）中。</li><li>注入2微升8毫毛地黄皂苷（8微米）到含有细胞悬浮液的oxygraph腔和透化细胞5分钟。 </li><li>注入的0.8M的苹果酸（5毫摩尔）12.5微升和10微升的2M的谷氨酸（10毫米）到oxygraph室中。记录细胞呼吸，直到稳定的氧通量信号得以实现。 </li><li>注入10微升的0.5M ADP（2.5毫摩尔）到oxygraph室并记录细胞呼吸，直到氧通量信号增加和稳定。 注意:ADP的添加到细胞会诱发在氧消耗的增加和氧通量信号将增加。 </li><li>注射2微升0.2毫米鱼藤酮（0.2μM）"小心"进入oxygraph室，并记录细胞呼吸，直到氧气流量信号下降并稳定。 </li><li>随后，注入20微升1M的琥珀酸酯（10毫米）进入oxygraph室，并记录细胞呼吸，直到氧气流量信号增加并稳定。 </li><li>然后，注入2微升5毫米的抗霉素A（5微米）"注意"到oxygraph室，并记录细胞呼吸，直到氧气流量信号下降并稳定。 </li><li>然后注入2.5微升0.8毫抗坏血酸（1毫摩尔），并立即注入后2.5微升0.2mM的TMPD（0.25毫摩尔）进oxygraph室并记录细胞呼吸，直到氧通量信号增加和稳定。 </li><li>最后注入10微升的1M叠氮化钠（5毫摩）&#39;小心"的入oxygraph室并记录细胞呼吸，直到氧通量信号下降并稳定。 </li></ol> 8.完整细胞耗氧<ol><li>以下步骤4.1到协议4.3中描述的方法制备的含oxygraph腔细胞悬浮液（1×10 6 / ml）中。 </li><li>注入1微升4毫克/毫升寡（2微克/毫升）装入含有oxygraph腔细胞悬浮液的D记录细胞呼吸，直到稳定的氧通量信号得以实现。 </li><li>随后注入1微升0.2毫米FCCP（0.1μM）"小心"进入oxygraph室，并记录细胞呼吸，直到氧气流量信号增加和稳定。 </li><li>注3微升0.2毫米FCCP（0.4微米）到oxygraph室，并进一步记录细胞呼吸，直到氧气流量信号增加和稳定。 </li><li>通过注入1-3微升0.2至1mM FCCP（0.1至2微米的最终浓度在腔室）插入oxygraph室，直到氧通量信号达到其最大水平，并没有进一步增加，然后滴定在0.1至0.3微米的步骤FCCP开始下降。 注:停止时氧信号达到最高水平，并开始下降，注射FCCP。 </li><li>然后，注入2微升0.2mM的鱼藤酮（0.2μM）和2微升的5mM的抗霉素A（5微米）到腔室的。记录呼吸，直至时间T他氧气流量信号下降并稳定。 </li></ol>

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The authors have nothing to disclose.

本议定书的目标是利用高分辨率的呼吸测量，测量线粒体呼吸链复合物"（I-IV）呼吸频率，最大线粒体电子传递系统的容量和线粒体外膜的完整性。 还有，本协议中的一些关键步骤。第一，蜂窝式氧消耗率通常标准化为细胞数目（皮摩尔/ [细胞秒×号]）。因此，监测蜂窝氧消耗之前，重要的是使用的设备，使细胞的数目的精确和可靠的测量。在本协议的自动细胞计数器已使用...

线粒体履行细胞能量代谢的重要作用，特别是通过使用氧气产生三磷酸腺苷（ATP）。它们被牵连的细胞死亡，并在多种人类疾病。线粒体氧化磷酸化（OXPHOS）结合一起耗氧量和ATP合成的电子传输链电子传递。线粒体三羧酸（TCA）循环涉及的蛋白质，碳水化合物和脂肪转化为能量丰富化合物作为烟酰胺腺嘌呤二核苷酸（NADH）和黄素腺嘌呤二核苷酸（FADH 2）。 （I至IV）位于线粒体内膜则NADH和FADH 2的电子被转移到呼吸电子传递链蛋白复合物。此外，另外两个氧化还原途径可以转移电子至电子传输链:ⅰ）线粒体电子转移黄素蛋白（ETF），其位于内线粒体的基质面从脂肪酸β氧化chondrial膜，和供给电子;及ii）线粒体甘油脱氢酶氧化甘油至磷酸二羟丙酮和线粒体电子传递链馈送电子。复合物IV（最终电子受体）的电子传送到一个氧分子，转换氧两份水分子。从呼吸电子传递链复合体I的电子的移动至IV是加上从线粒体基质以它建立跨线粒体内膜的电化学梯度的间空间质子流量。事后，线粒体复合物V（ATP合酶）穿梭氢离子放回线粒体基质并合成ATP分子。 OXPHOS函数可以在体内和体外使用各种技术和各种线粒体呼吸状态可以得到进行评估。在分离线粒体以下respiratoRY状态可测:ⅰ）基底线粒体呼吸（状态1）在加入线粒体呼吸链复合物的特异性底物的后，ⅱ）氧气量（状态2），ⅲ）最大线粒体耗氧量加入饱和浓度的后腺苷二磷酸（ADP）（状态3）和，ⅳ）ADP量（转化为ATP后搁置呼吸）（状态4）。在完整细胞以下呼吸状态可测:ⅰ）基底蜂窝耗氧内源性底物和ADP的存在下，ⅱ）在寡霉素的存在下基底蜂窝氧消耗（寡霉素不敏感的呼吸）和寡敏感呼吸（ATP营业额），ⅲ）FCCP解偶联呼吸作用和加法抗霉素A和鱼藤酮的后ⅳ）非线粒体呼吸。在透化的细胞中，电子传递链复合物和ADP的特异性底物，可以添加和最大复杂依赖性呼吸率小号UCH复杂I-，II-和IV依赖性呼吸率可以被测量。 细胞呼吸的测量提供了重要的见解特定络合物I-IV，线粒体完整性和能量代谢1，2，3线粒体呼吸的能力。其中之一使线粒体耗氧具有高精确度，分辨率和灵敏度的测量装置是高分辨率oxygraph 4。高分辨率oxygraph设备包含两个室与喷射口和每个室配备有极谱氧传感器。蜂窝式或分离的线粒体悬浮液中的呼吸连续搅拌。为了评估线粒体功能，底物和抑制剂对线粒体复合物活性可以根据标准协议来添加。底物和抑制剂可通过注射INT滴定澳oxygraph的内室，耗氧率正在使用的软件计算并表示为每细胞数第二皮摩尔。高分辨率呼​​吸测量比传统和常规极谱氧电极装置，包括增加的敏感性，并与生物样品如完整的或透化的细胞的小的数字工作能力提供了几个优点。此外，每个设备包括两个腔室，和呼吸率可以同时记录的氧浓度的比较。高分辨率oxygraph还具有相对于传统极谱氧电极装置从设备室的氧减少了泄漏的优点。最近开发的用于测量蜂窝氧消耗另一个设备是96孔细胞外磁通分析器5。胞外通量分析仪配有荧光代替极谱法传感器。外的优点因此磁通分析器相比高分辨率oxygraph是ⅰ）它是一个高度自动化设备，ii）其可以测量在24和用于高通量筛选96孔板耗氧量，需要较低量的生物样品，和iii）细胞糖酵解流量的附加测量是可能的。胞外通量分析仪相比于高分辨率oxygraph的缺点是:i）所述设备和消耗品如荧光板，这是不可再用的成本高，和ii）每测定只有四种化合物/孔是可注射因此该系统是不为衬底解偶联剂抑制剂滴定协议可行的。 在本研究中，我们使用高分辨率的呼吸测量来确定线粒体呼吸。用于蜂窝耗氧量的实验中，将细胞透化，以允许外源ADP和可氧化线粒体基质的入口用于供给电子到配合物S中的呼吸系统。这种方法允许个别线粒体络合物呼吸能力夹层，这是一个明显的优势相比完整细胞（多底物是细胞不通透性）。然而，细胞膜透化会破坏细胞质和细胞外的空间和介质（洗出胞质溶质）和细胞内空间的组合物之间的屏障与胞外介质平衡。一个透化的细胞在完整细胞的缺点之一是，如果细胞透期间采用洗涤剂过量线粒体外膜可被损坏。在完整细胞，基底，完整细胞的耦合和解偶联呼吸作用可以测量。这种方法计算的完整细胞的氧消耗而不添加外源性底物和ADP的，在一体化细胞再现呼吸功能并且还提供对最大线粒体电子TRANSPO信息室温容量6,7。之一的完整细胞的过度渗透细胞的优点是，蜂窝环境将不中断，线粒体是在与细胞的整个组分的接触。为了透化细胞质膜，洗涤剂如毛地黄皂苷已用于8。然而，如果采用毛地黄皂苷过量线粒体外膜的完整性可受到损害。以确认线粒体外膜的完整性在透化的细胞不受损害，毛地黄皂苷滴定被执行以确定用于蜂窝透的最佳浓度。对于这些实验，将细胞再悬浮于呼吸介质和毛地黄皂苷浓度由线粒体底物和ADP和呼吸速率的存在呼吸测量滴定进行测定。完好，无渗透细胞的呼吸是不mitocho的存在刺激ndrial基板和ADP。然而，随后逐步毛地黄皂苷滴定将产生等离子体的膜逐渐透，并且获得最佳的毛地黄皂苷浓度。这是由呼吸达到充分透的增加所示。线粒体的质量和外膜的完整性可以通过添加外源细胞色素C 2,9进行验证。细胞色素c是线粒体电子传递链10，11，12的12 kDa的电子携载蛋白。它被定位于线粒体膜间隙，并参与耗氧量，携带来自复合物III电子以复合物IV。一旦线粒体外膜被破坏，细胞色素C释放和线粒体氧消耗减少。在加入外源细胞色素c，在线粒体呼吸任何增强指示的一个破坏线粒体外膜。 在透细胞，基质和线粒体复合体活性的抑制剂加入以下各种协议3,9。例如为了研究线粒体复合驱动呼吸速率，可以使用以下协议。细胞的透化后，第一复合物I由基板苹果酸盐和谷氨酸盐，其中生成的NADH作为底物的呼吸链和挑衅复杂导之后，ADP加到转换为ATP（状态3，活性的活化刺激的复杂的I-依赖呼吸）。达到稳定的信号后，鱼藤酮（线粒体复合物I抑制剂）被施用以抑制复杂一鱼藤酮后跟琥珀到FADH 2和激活复合体II（状态3，活性复合物II依赖呼吸）。为了测量复合物IV依赖性呼吸，第一复合物III依赖呼吸是通过添加抗霉素A（线粒体复合物III抑制剂）抑制。之后，复合物IV依赖性呼吸通过施用抗坏血酸和tetramethylphenylendiamine（TMPD）刺激。在呼吸缓冲TMPD可自动氧化，因此最大复合物IV依赖性呼吸速率（状态3）由之前和加入叠氮化钠，线粒体复合物IV的抑制剂后减去呼吸速率计算。呼吸实验可以在平行酮作为对照（未刺激细胞）的oxygraph的两个腔室，另一个含有刺激的细胞来进行。显然，细胞可以以各种方式， 例如 ，预先处理，以影响线粒体功能的药物，在oxygraph室被测量它们的氧气消耗量之前。该协议允许个体线粒体呼吸链复合物的功能检查。此外，一个可以测量最大ADP刺激呼吸通透细胞，棕榈酸的形式使用外源性脂肪酸（状态3）。在这个协议中，棕榈酸钠的浓缩存量缀合的超无脂肪酸的牛血清白蛋白（BSA）（6:1摩尔比棕榈酸:BSA）中。之后，将细胞首先被透用毛地黄皂苷和线粒体呼吸通过加入肉碱评估和棕榈酸，随后加入ADP（状态3，最大呼吸）的。然后，寡霉素加到模拟状态4（状态40），并呼吸控制率（RCR值）计算为状态3 /状态4O。 β氧化促进生产乙酰CoA（其进入在TCA循环），并代FADH 2和NADH，电子内容通过电子转移黄素蛋白和β羟酰-CoA脱氢酶传递到电子传递链。线粒体是在脂肪酸代谢的中心与所述棕榈酸BSA的协议可以由研究者检查脂肪可以使用TY酸氧化。在完整细胞，活化和线粒体络合物活性的抑制剂加入以下一个不同的协议6,9。对于这些实验，在不存在外源性底物的非透化的细胞的第一氧消耗测量（磷酸呼吸率）。然后，所述非磷酸化的呼吸速率在加入寡的，这是线粒体ATP合酶的抑制剂后进行测定。此后，protonophore羰氰化物对 - trifluoromethoxyphenylhydrazone（FCCP）以各种浓度被施用并测量最大线粒体耦合呼吸速率。 Protonophores如FCCP可诱导内膜的质子透过性的增大，使质子的被动运动消散化学渗透梯度。质子通透性增加氧化脱开呼吸（无ATP生产），并诱导邻增加xygen消耗。之后，鱼藤酮和抗霉素A加到抑制线粒体呼吸和非线粒体呼吸从所有其他呼吸率中减去。 氧消耗率可以表示为其中是通过分割特定体积的氧通量（在闭合氧舱）计算值IO 2 [皮摩尔点¯x秒-1×10 -6细胞（每百万个细胞的氧气流量），N-，O- 2 [皮摩尔点¯x秒到1×毫升-1]通过细胞浓度在细胞室（细胞每卷号码[10 6个细胞∙毫升1]）15。小区质量比氧通量，JO 2 [皮摩尔点¯x秒到1×毫克-1]，是每信元流，IO 2 [皮摩尔点¯x秒-1×10 -6细胞]，以质量每个小区划分[毫克∙10 ] 6细胞;或体积比通量，合资，O 2 [皮摩尔点¯x秒到1×毫升-1]，每卷分为质量乌梅[毫克∙毫升1]。 JO 2是每细胞蛋白，干重或细胞体积的氧通量。 在使用高分辨率呼​​吸测量本研究中，我们描述了协议使用外源性细胞色素C来确定i）在完整细胞质膜通透性最佳毛地黄皂苷浓度（毛地黄皂苷滴定测定），ⅱ）线粒体外膜的完整性，ⅲ）线粒体呼吸链复合物我，在外源ADP和线粒体呼吸链底物，和存在毛地黄皂苷通透HepG2细胞II和IV最大呼吸率ⅳ）基，耦合和完整细胞的最大解偶联呼吸作用（最大电子传输容量）不加入外源基片的和ADP，再现中的一体化细胞的呼吸功能。

<table><tbody><tr><td>ADP</td><td>Sigma</td><td>A 4386</td><td>Chemical</td></tr><tr><td>Antimycin A</td><td>Sigma</td><td>A 8674</td><td>Chemical, dissolve in ethanol</td></tr><tr><td>Ascorbate</td><td>Merck</td><td>1.00127</td><td>Chemical</td></tr><tr><td>BSA</td><td>Sigma</td><td>A 6003</td><td>Chemical</td></tr><tr><td>FCCP</td><td>Sigma</td><td>C 2920</td><td>Chemical, dissolve in ethanol</td></tr><tr><td>Countess automated cell counter&amp;nbsp;</td><td>Thermo Fisher Scientific</td><td>n/a</td><td>Automated cell counting instrument</td></tr><tr><td>Cytochrome c</td><td>Sigma</td><td>C 7752</td><td>Chemical</td></tr><tr><td>Digitonin</td><td>Sigma</td><td>D 5628</td><td>Chemical, dissolve in DMSO</td></tr><tr><td>DMEM</td><td>Gibco</td><td>31966021</td><td>Medium</td></tr><tr><td>EGTA</td><td>fluka</td><td>3779</td><td>Chemical</td></tr><tr><td>FBS</td><td>Gibco</td><td>26010-074</td><td>Medium component</td></tr><tr><td>Glutamate</td><td>Sigma</td><td>G 1626</td><td>Chemical</td></tr><tr><td>Hepes</td><td>Sigma</td><td>H 7523</td><td>Chemical</td></tr><tr><td>KCl</td><td>Merck</td><td>1.04936</td><td>Chemical</td></tr><tr><td>KH&lt;sub&gt;2&lt;/sub&gt;PO&lt;sub&gt;4&lt;/sub&gt;</td><td>Merck</td><td>1.04873</td><td>Chemical</td></tr><tr><td>K-lactobionate</td><td>Sigma</td><td>L 2398</td><td>Chemical</td></tr><tr><td>MgCl&lt;sub&gt;2&lt;/sub&gt;</td><td>Sigma</td><td>M 9272</td><td>Chemical</td></tr><tr><td>O2k-Core: Oxygraph-2k&amp;nbsp;</td><td>Oroboros Instruments</td><td>10000-02</td><td>High-resolution respirometry instrument</td></tr><tr><td>Oligomycin</td><td>Sigma</td><td>O 4876</td><td>Chemical, dissolve in ethanol</td></tr><tr><td>Penicillin-streptomycin</td><td>Gibco</td><td>15140122</td><td>Chemical</td></tr><tr><td>Sodium azide</td><td>Sigma</td><td>S2002</td><td>Chemical</td></tr><tr><td>Rotenone</td><td>Sigma</td><td>R 8875</td><td>Chemical, dissolve in ethanol</td></tr><tr><td>Succinate</td><td>Sigma</td><td>S 2378</td><td>Chemical</td></tr><tr><td>Taurine</td><td>Sigma</td><td>T 8691</td><td>Chemical</td></tr><tr><td>TMPD</td><td>Sigma</td><td>T 3134</td><td>Chemical</td></tr><tr><td>Trypsin</td><td>Sigma</td><td>T 4674</td><td>Chemical</td></tr></tbody></table>

high-resolution respirometry to assess mitochondrial function in permeabilized and intact cells

A high-resolution oxygraph is a device for measuring cellular oxygen consumption in a closed-chamber system with very high resolution and sensitivity in biological samples (intact and permeabilized cells, tissues or isolated mitochondria). The high-resolution oxygraph device is equipped with two chambers and uses polarographic oxygen sensors to measure oxygen concentration and calculate oxygen consumption within each chamber. Oxygen consumption rates are calculated using software and expressed as picomoles per second per number of cells. Each high-resolution oxygraph chamber contains a stopper with injection ports, which makes it ideal for substrate-uncoupler-inhibitor titrations or detergent titration protocols for determining effective and optimum concentrations for plasma membrane permeabilization. The technique can be applied to measure respiration in a wide range of cell types and also provides information on mitochondrial quality and integrity, and maximal mitochondrial respiratory electron transport system capacity.

最佳毛地黄皂苷含量的测定细胞透:毛地黄皂苷滴定实验毛地黄皂苷滴定被执行以确定用于HepG2细胞透化的最佳浓度。对于这些实验，毛地黄皂苷是在完整细胞滴定在鱼藤酮，琥珀酸盐（线粒体复合物II衬底）和ADP的饱和量的存在下，和呼吸率在基线和之后每个测量（到复合体II依赖性状态3诱导）滴定（ 图1A和1B?...

Watch this Scientific Journal Video about High-resolution Respirometry to Assess Mitochondrial Function in Permeabilized and Intact Cells at JoVE.com

高分辨率呼​​吸测定法评估透化和完整细胞中的线粒体功能生物学 |视频

High-resolution Respirometry to Assess Mitochondrial Function in Permeabilized and Intact Cells

高分辨率呼​​吸测量被用于确定线粒体耗氧量。这是一个简单的技术来确定线粒体呼吸链复合物&#39;（I-IV）的呼吸率，最大线粒体电子传递系统容量，和线粒体外膜的完整性。

高分辨率呼​​吸测量，以评估线粒体功能的通透和完整细胞

A high-resolution oxygraph is a device for measuring cellular oxygen consumption in a closed-chamber system with very high resolution and sensitivity in ...

high-resolution-respirometry-to-assess-mitochondrial-function

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41.3K 次观看.  University of Bern. 高分辨率呼​​吸测量被用于确定线粒体耗氧量。这是一个简单的技术来确定线粒体呼吸链复合物'（I-IV）的呼吸率，最大线粒体电子传递系统容量，和线粒体外膜的完整性。 

视频: High-resolution Respirometry to Assess Mitochondrial Function in Permeabilized and Intact Cells

Assessing Mitochondrial Function in Sciatic Nerve by High-Resolution Respirometry

Mitochondrial dysfunction in peripheral nerves accompanies several diseases associated with peripheral neuropathy, which can be triggered by multiple causes, including autoimmune diseases, diabetes, infections, inherited disorders, and tumors. Assessing mitochondrial function in mouse peripheral nerves can be challenging due to the small sample size, a limited number of mitochondria present in the tissue, and the presence of a myelin sheath. The technique described in this work minimizes these challenges by using a unique permeabilization protocol adapted from one used for muscle fibers, to assess sciatic nerve mitochondrial function instead of isolating the mitochondria from the tissue. By measuring fluorimetric reactive species production with Amplex Red/Peroxidase and comparing different mitochondrial substrates and inhibitors in saponin-permeabilized nerves, it was possible to detect mitochondrial respiratory states, reactive oxygen species (ROS), and the activity of mitochondrial complexes simultaneously. Therefore, the method presented here offers advantages compared to the assessment of mitochondrial function by other techniques.

High-resolution respirometry coupled to fluorescence sensors determines mitochondrial oxygen consumption and reactive oxygen species (ROS) generation. The present protocol describes a technique to assess mitochondrial respiratory rates and ROS production in the permeabilized sciatic nerve.

通过高分辨率呼吸测量法评估坐骨神经的线粒体功能

Mitochondrial dysfunction in peripheral nerves accompanies several diseases associated with peripheral neuropathy, which can be triggered by multiple ...

科研

生物化学

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

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

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

线粒体的制备及透气性评估，从骨骼肌组织通过穿刺活检获得的分离

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

医学

Multi-parameter Measurement of the Permeability Transition Pore Opening in Isolated Mouse Heart Mitochondria

The mitochondrial permeability transition pore (mtPTP) is a non specific channel that forms in the inner mitochondrial membrane to transport solutes with a molecular mass smaller than 1.5 kDa. Although the definitive molecular identity of the pore is still under debate, proteins such as cyclophilin D, VDAC and ANT contribute to mtPTP formation. While the involvement of mtPTP opening in cell death is well established1, accumulating evidence indicates that the mtPTP serves a physiologic role during mitochondrial Ca2+ homeostasis2, bioenergetics and redox signaling 3.
mtPTP opening is triggered by matrix Ca2+ but its activity can be modulated by several other factors such as oxidative stress, adenine nucleotide depletion, high concentrations of Pi, mitochondrial membrane depolarization or uncoupling, and long chain fatty acids4. In vitro, mtPTP opening can be achieved by increasing Ca2+ concentration inside the mitochondrial matrix through exogenous additions of Ca2+ (calcium retention capacity). When Ca2+ levels inside mitochondria reach a certain threshold, the mtPTP opens and facilitates Ca2+ release, dissipation of the proton motive force, membrane potential collapse and an increase in mitochondrial matrix volume (swelling) that ultimately leads to the rupture of the outer mitochondrial membrane and irreversible loss of organelle function.
Here we describe a fluorometric assay that allows for a comprehensive characterization of mtPTP opening in isolated mouse heart mitochondria. The assay involves the simultaneous measurement of 3 mitochondrial parameters that are altered when mtPTP opening occurs: mitochondrial Ca2+ handling (uptake and release, as measured by Ca2+ concentration in the assay medium), mitochondrial membrane potential, and mitochondrial volume. The dyes employed for Ca2+ measurement in the assay medium and mitochondrial membrane potential are Fura FF, a membrane impermeant, ratiometric indicator which undergoes a shift in the excitation wavelength in the presence of Ca2+, and JC-1, a cationic, ratiometric indicator which forms green monomers or red aggregates at low and high membrane potential, respectively. Changes in mitochondrial volume are measured by recording light scattering by the mitochondrial suspension. Since high-quality, functional mitochondria are required for the mtPTP opening assay, we also describe the steps necessary to obtain intact, highly coupled and functional isolated heart mitochondria.

A spectrofluorometric protocol for the measurement of the mitochondrial permeability transition pore opening in isolated mouse heart mitochondria is presented here. The assay involves the simultaneous measurement of mitochondria Ca2+ handling, mitochondrial membrane potential and mitochondrial volume. The procedure for obtaining high-quality and functional heart mitochondria is also described.

小鼠离体心肌线粒体通透性转换孔开放中的多参数测量。

The mitochondrial permeability transition pore (mtPTP) is a non specific channel that forms in the inner mitochondrial membrane to transport solutes with ...

生物学

Isolation of Intact Mitochondria from Skeletal Muscle by Differential Centrifugation for High-resolution Respirometry Measurements

Mitochondria are involved in cellular energy metabolism and use oxygen to produce energy in the form of adenosine triphosphate (ATP). Differential centrifugation at low- and high-speed is commonly used to isolate mitochondria from tissues and cultured cells. Crude mitochondrial fractions obtained by differential centrifugation are used for respirometry measurements. The differential centrifugation technique is based on the separation of organelles according to their size and sedimentation velocity. The isolation of mitochondria is performed immediately after tissue harvesting. The tissue is immersed in an ice-cold homogenization medium, minced using scissors and homogenized in a glass homogenizer with a loose-fitting pestle. The differential centrifugation technique is efficient, fast and inexpensive and the mitochondria obtained by differential centrifugation are pure enough for respirometry assays. Some of the limitations and disadvantages of isolated mitochondria, based on differential centrifugation, are that the mitochondria can be damaged during the homogenization and isolation procedure and that large amounts of the tissue biopsy or cultured cells are required for the mitochondrial isolation.

Here, a quadriceps muscle specimen is taken from an anaesthetized pig and mitochondria are isolated by differential centrifugation. Then, the respiratory rates of mitochondrial respiratory chain complexes I, II and IV are determined using high-resolution respirometry.

骨骼肌完整的线粒体的分离差速离心高分辨率测量呼吸运动

Mitochondria are involved in cellular energy metabolism and use oxygen to produce energy in the form of adenosine triphosphate (ATP). Differential ...

High-resolution Respirometry to Measure Mitochondrial Function of Intact Beta Cells in the Presence of Natural Compounds

High-resolution respirometry allows for the measurement of oxygen consumption of isolated mitochondria, cells and tissues. Beta cells play a critical role in the body by controlling blood glucose levels through insulin secretion in response to elevated glucose concentrations. Insulin secretion is controlled by glucose metabolism and mitochondrial respiration. Therefore, measuring intact beta cell respiration is essential to be able to improve beta cell function as a treatment for diabetes. Using intact 832/13 INS-1 derived beta cells we can measure the effect of increasing glucose concentration on cellular respiration. This protocol allows us to measure beta cell respiration in the presence or absence of various compounds, allowing one to determine the effect of given compounds on intact cell respiration. Here we demonstrate the effect of two naturally occurring compounds, monomeric epicatechin and curcumin, on beta cell respiration under the presence of low (2.5 mM) or high glucose (16.7 mM) conditions. This technique can be used to determine the effect of various compounds on intact beta cell respiration in the presence of differing glucose concentrations.

The goal of this protocol is to measure the effect of glucose-mediated changes to mitochondrial respiration in the presence of natural compounds on intact 832/13 beta cells using high-resolution respirometry.

在天然化合物存在下测定完整β细胞线粒体功能的高分辨率呼吸

High-resolution respirometry allows for the measurement of oxygen consumption of isolated mitochondria, cells and tissues. Beta cells play a critical role ...

通过高分辨率呼吸测定法测定人精子细胞中的呼吸

High-Resolution Respirometry to Assess Mitochondrial Function in Human Spermatozoa

Semen quality is often studied by routine semen analysis, which is descriptive and often inconclusive. Male infertility is associated with altered sperm mitochondrial activity, so the measurement of sperm mitochondrial function is an indicator of sperm quality. High-resolution respirometry is a method of measuring the oxygen consumption of cells or tissues in a closed-chamber system. This technique can be implemented to measure respiration in human sperm and provides information about the quality and integrity of the sperm mitochondria. High-resolution respirometry allows the cells to move freely, which is an a priori advantage in the case of sperm. This technique can be applied with intact or permeabilized spermatozoa and allows for the study of intact sperm mitochondrial function and the activity of individual respiratory chain complexes. The high-resolution oxygraph instrument uses sensors to measure the oxygen concentration coupled with sensitive software to calculate the oxygen consumption. The data are used to calculate respiratory indices based on the oxygen consumption ratios. Consequently, the indices are the proportions of two oxygen consumption rates and are internally normalized to the cell number or protein mass. The respiratory indices are an indicator of sperm mitochondrial function and dysfunction.

作者聚焦:通过揭示精子代谢和线粒体功能推进男性不育研究 

The analysis of sperm mitochondrial function by high-resolution respirometry permits the measurement of the oxygen consumption of freely moving spermatozoa in a closed-chamber system. The technique can be applied to measure respiration in human spermatozoa, which provides information on sperm mitochondrial characteristics and integrity.

高分辨率呼吸测定法评估人类精子的线粒体功能

Semen quality is often studied by routine semen analysis, which is descriptive and often inconclusive. Male infertility is associated with altered sperm ...

Measuring Mitochondrial Substrate Flux in Recombinant Perfringolysin O-Permeabilized Cells

Mitochondrial substrate flux is a distinguishing characteristic of each cell type, and changes in its components such as transporters, channels, or enzymes are involved in the pathogenesis of several diseases. Mitochondrial substrate flux can be studied using intact cells, permeabilized cells, or isolated mitochondria. Investigating intact cells encounters several problems due to simultaneous oxidation of different substrates. Besides, several cell types contain internal stores of different substrates that complicate results interpretation. Methods such as mitochondrial isolation or using permeabilizing agents are not easily reproducible. Isolating pure mitochondria with intact membranes in sufficient amounts from small samples is problematic. Using non-selective permeabilizers causes various degrees of unavoidable mitochondrial membrane damage. Recombinant perfringolysin O (rPFO) was offered as a more appropriate permeabilizer, thanks to its ability to selectively permeabilize plasma membrane without affecting mitochondrial integrity. When used in combination with microplate respirometry, it allows testing the flux of several mitochondrial substrates with enough replicates within one experiment while using a minimal number of cells. In this work, the protocol describes a method to compare mitochondrial substrate flux of two different cellular phenotypes or genotypes and can be customized to test various mitochondrial substrates or inhibitors.

In this work, we describe a modified protocol to test mitochondrial respiratory substrate flux using recombinant perfringolysin O in combination with microplate-based respirometry. With this protocol, we show how metformin affects mitochondrial respiration of two different tumor cell lines.

测量重组透气溶性细胞中线粒体底物通量 O-透化细胞

Mitochondrial substrate flux is a distinguishing characteristic of each cell type, and changes in its components such as transporters, channels, or ...

High-Resolution Respirometry to Assess Bioenergetics in Cells and Tissues Using Chamber- and Plate-Based Respirometers

High-resolution respirometry (HRR) allows monitoring oxidative phosphorylation in real-time for analysis of individual cellular energy states and assessment of respiratory complexes using diversified substrate-uncoupler-inhibitor titration (SUIT) protocols. Here, the usage of two high-resolution respirometry devices is demonstrated, and a basic collection of protocols applicable for the analysis of cultured cells, skeletal and heart muscle fibers, and soft tissues such as the brain and liver are presented. Protocols for cultured cells and tissues are provided for a chamber-based respirometer and cultured cells for a microplate-based respirometer, both encompassing standard respiration protocols. For comparative purposes, CRISPR-engineered HEK293 cells deficient in mitochondrial translation causing multiple respiratory system deficiency are used with both devices to demonstrate cellular defects in respiration. Both respirometers allow for comprehensive measurement of cellular respiration with their respective technical merits and suitability dependent on the research question and model under study.

Assessing oxidative phosphorylation using high-resolution respirometers has become an integral part of the functional analysis of mitochondria and cellular energy metabolism. Here, we present protocols for the analysis of cellular energy metabolism using chamber and microplate-based high-resolution respirometers and discuss the key benefits of each device.

高分辨率呼吸测量，以评估线粒体功能的通透和完整细胞

摘要

探索更多视频

此视频中的章节

通过高分辨率呼吸测量法评估坐骨神经的线粒体功能

线粒体的制备及透气性评估，从骨骼肌组织通过穿刺活检获得的分离

小鼠离体心肌线粒体通透性转换孔开放中的多参数测量。

骨骼肌完整的线粒体的分离差速离心高分辨率测量呼吸运动

在天然化合物存在下测定完整β细胞线粒体功能的高分辨率呼吸

高分辨率呼吸测定法评估人类精子的线粒体功能

高分辨率呼吸测定法评估人类精子的线粒体功能

高分辨率呼吸测定法评估人类精子的线粒体功能

测量重组透气溶性细胞中线粒体底物通量 O-透化细胞

高分辨率呼吸测定法，使用基于腔室和板式呼吸计评估细胞和组织中的生物能量学

高分辨率呼​​吸测量，以评估线粒体功能的通透和完整细胞

摘要

探索更多视频

此视频中的章节

通过高分辨率呼吸测量法评估坐骨神经的线粒体功能

线粒体的制备及透气性评估，从骨骼肌组织通过穿刺活检获得的分离

小鼠离体心肌线粒体通透性转换孔开放中的多参数测量。

骨骼肌完整的线粒体的分离差速离心高分辨率测量呼吸运动

在天然化合物存在下测定完整β细胞线粒体功能的高分辨率呼吸

高分辨率呼吸测定法评估人类精子的线粒体功能

高分辨率呼吸测定法评估人类精子的线粒体功能

高分辨率呼吸测定法评估人类精子的线粒体功能

测量重组透气溶性细胞中线粒体底物通量 O-透化细胞

高分辨率呼吸测定法，使用基于腔室和板式呼吸计评估细胞和组织中的生物能量学

高分辨率呼吸测量，以评估线粒体功能的通透和完整细胞