我们承认美国国立卫生研究院资助P30ES013508和T32ES019851的支持。

伦理学声明:关于人体标本的处理所有协议遵循美国宾夕法尼亚大学的人类研究伦理委员会的指导方针。 1.缓冲区的制备及100X母液<ol><li>制备1升的碱蒂罗德缓冲液中。结合8.123克氯化钠，1.428克碳酸氢钠，0.466克氯化钙 ∙2 H 2 0，0.224克KCl和0.095克的MgCl 2，调整总体积至1L用的DDH 2 O过滤消毒基地台氏缓冲区。 </li><li>准备葡萄糖和棕榈酸的100倍储备溶液。对于100倍的葡萄糖储备液，溶解100毫克的葡萄糖或[13 C 6]葡萄糖1毫升的DDH 2 O的为100倍，棕榈酸溶液，溶解2.56毫克棕榈酸或在1ml乙醇2.72毫克[13 C 16] -棕榈酸。过滤消毒100X原液。 </li><li>准备适当富集台氏缓冲器<ol&gt; <li>对于非标记研究，加入1毫升的100倍的葡萄糖储备溶液和1ml的100倍的棕榈酸储备溶液至98的基台氏缓冲毫升。 </li><li>葡萄糖标记研究，加入1毫升的100×[13 C 6]葡萄糖储备溶液和1ml的100倍的棕榈酸储备溶液至98的基台氏缓冲毫升。 </li><li>为棕榈酸标记研究，将1ml [13 C 16] -棕榈酸原液的100倍葡萄糖储备溶液和1ml添加到98的基台氏缓冲毫升。 </li></ol></li><li>准备鱼藤酮治疗解决方案<ol><li>制备鱼藤酮在二甲亚砜（DMSO）中的10mM储液。 </li><li>对于非贴标剂量反应的研究，进行1.4.3。对于以单剂量标记研究，继续执行1.4.5。 </li><li>在DMSO执行的10mM鱼藤酮的库存的系列稀释以获得具有鱼藤酮的浓度范围为1nM至100μM的解决方案。这是100倍的股票秒。 </li><li>加入50微升每只股票的5毫升基地台氏缓冲+葡萄糖+棕榈酸从1.3.1准备用鱼藤酮浓度从晚上10点到1微米的解决方案。备选地，添加50微升的DMSO作为载体对照。继续执行2.1。 </li><li>稀释的10mM储备液1,000倍的DMSO以获得10微米的工作溶液。 </li><li>加入50微升这股给每个基地台氏缓冲+5毫升[13 C 6]葡萄糖+棕榈酸从1.3.2和基地台氏缓冲+葡萄糖+ [13 C 16]从1.3.3 -棕榈酸。的最终浓度为100nM的鱼藤酮。另外，加入50微升DMSO的每一个缓冲区，作为车辆的控制。 </li></ol></li></ol> 2.血小板隔离注意:此方法是适合于从全血或血小板从衍生袋血小板。本文中的示例数据准备利用血小板衍生袋血小板。请参见Basu等人 5为使用从全血中分离血小板有关的更多细节。 <ol><li>从全血中分离血小板 注意:如果从血小板一袋血小板分离，继续2.2.1。 <ol><li>离心全血为在没有刹车的15分钟175×g的。 </li><li>传输1毫升上层富血小板血浆（PRP）层到1.5ml微量离心管中。 </li><li>离心在400×g下将PRP 5分钟。 </li><li>吸上清并继续执行步骤3.1或3.2。 </li></ol></li><li>从血小板袋血小板隔离<ol><li>传输1毫升血小板悬浮液至1.5ml微量离心管中。 </li><li>离心在400 xg离心悬浮液5分钟。 </li><li>吸上清并继续执行步骤3.1。或3.2。 </li></ol></li></ol> 3.进行实验<ol><li>为了确定异生的作用治疗（ 例如，鱼藤酮）上感兴趣的代谢物的水平（ 例如，乙酰-CoA），继续3.1.1。来确定到感兴趣的下游代谢产物的代谢前体的掺入生物外源性（ 例如 ，鱼藤酮）的效果，进行3.2.1。 <ol><li>不使用少于三个生物复制为每个条件，从步骤2.1.4或2.2.3在1毫升每种解决方案的步骤1.4.4悬浮颗粒血小板。 </li><li>在水夹套的 CO 2培养箱设定为95％湿度，5％CO 2和37℃下1小时孵育再悬浮血小板。继续执行步骤4.1 注意:这里介绍一个剂量反应实验。或者，时程实验可以做，其中所述剂量是固定和温育的长度是变化的，而不是。 </li></ol></li><li>以确定代谢前体掺入的生物外源性处理的效果到感兴趣的下游代谢产物。 <ol><li> ü唱不超过每个条件的三次生物学重复较少，从步骤2.1.4或2.2.3在标台氏缓冲每个配方1毫升步骤1.4.6悬浮颗粒血小板。 </li><li>在水夹套的 CO 2培养箱设定为95％湿度，5％CO 2和37℃下1小时孵育再悬浮血小板。继续执行步骤4.1。 </li></ol></li></ol> 4.淬火和CoA提取<ol><li>通过以3000 xg离心离心3分钟沉淀血小板。 </li><li>吸上清。 </li><li>在750微升冰冷的10％三氯乙酸悬浮血小板（重量/体积）。 </li><li>添加适当的稳定同位素标记的内部标准。例如，如果目标是辅酶物种的水平跨样品比较，用100微升的稳定同位素如前所述从酵母得到的标辅酶A硫酯（SILEC技术）29的混合物。 </li><li>脉冲声处理每个样品以0.5秒的脉冲30​​次。 </li><li>离心样品以16,000 xg离心在4℃下10分钟。 </li><li>贴上C18固相萃取（SPE）列真空歧管。 </li><li>调节用1ml甲醇的列。 </li><li>平衡柱用1ml的DDH 2 O </li><li>通过列运行样品衍生的上清液（约850微升在这种情况下）。 </li><li>用1毫升清水洗净列。 </li><li>放入10毫升玻璃离心管到真空歧管，收集洗脱部分。 </li><li>用在甲醇中加入1ml的25mM乙酸铵洗脱该柱。 </li><li>在氮气下干燥洗脱液。 </li><li>悬浮干燥残留在50μl5％（重量/体积）的5-磺基水杨酸并转移到HPLC小瓶中。 </li></ol> 5. HPLC安装<ol><li>使用控制软件的HPLC系统中，创建与目标分析物和利用柱进行了优化，如表1中所列的HPLC条件的方法。 注:列TEMPERAT用于该数据的生成URE为25℃，但在特定的参数将通过仪器和相对于感兴趣的化合物而变化。为酰基辅酶A的定量，LC-MS分析的多种方法已被报道，并验证在不同LC条件14,19,20不同的分析物。 </li><li>让HPLC充分平衡。 注意:这应包括溶剂的两个涂刷在该方法的起始溶剂条件附接柱的柱和平衡之前。该平衡容积应按照制造商的指示，以及由此产生的污水应分流到废液。 注:请巴苏和布莱尔5关于HPLC设置的详细信息。 </li></ol> 6.质谱仪设置<ol><li>使用控制软件用于质谱仪，创建用于检测感兴趣的化合物的离子化形式靶向方法。参数介绍在表2中。 注意:这些化合物的稳定同位素类似物应被用来确定一个正或负模式下是否能给予优良的灵敏度和优化源和光学条件为它们的检测。质谱仪方法的总检测时间应该对应于相关的高效液相色谱法的时间分量。 注意:如前面提到的，酰基-CoA分析的多种方法已被报道，包括正面14和负电离15模式和使用高分辨率质谱仪17的，而不是一个三重四极杆仪器28。 </li><li>清洁和校准仪器，建立一个稳定的喷雾进入光谱仪，并且允许时间校准溶液充分从源，并根据制造商的说明光学消散。 </li><li>设置为所有与质谱仪控制软件的样本的序列。 Inclu取消名称或数字标识符，并注明相应的注射量和仪器法。运行的第一个样品之前的空白，并根据需要来减轻残留或基体效应样品之间运行的其他空白和洗涤。 注意:峰积分所选分析物"液体色谱的处理方法，通常可以此顺序内设定或随后在数据处理中应用。 </li><li>开始的顺序和周期性地监视质谱仪和HPLC系统的问题，包括压力波动或系统故障以及在运​​行期间的信号强度的损失。 注:严重问题或持续运行故障可能需要停止运行和吹扫和重新平衡的HPLC系统以及清洁和校准质谱仪。巴苏咨询和布莱尔5关于MS的设置和数据处理的更多细节。 </li></ol>

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Biochem. 474, 59-65 (2015).</li><li>Wallace, D. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mitochondrial+diseases+in+man+and+mouse.">Mitochondrial diseases in man and mouse.</a> Science. 283 (5407), 1482-1488 (1999).</li><li>Wojtovich, A. P., Brookes, P. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+complex+II+inhibitor+atpenin+A5+protects+against+cardiac+ischemia-reperfusion+injury+via+activation+of+mitochondrial+KATP+channels.">The complex II inhibitor atpenin A5 protects against cardiac ischemia-reperfusion injury via activation of mitochondrial KATP channels.</a> Basic Res. Cardiol. 104 (2), 121-129 (2009).</li><li>Worth, A. 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=Stable+isotopes+and+LC-MS+for+monitoring+metabolic+disturbances+in+Friedreich's+ataxia+platelets.">Stable isotopes and LC-MS for monitoring metabolic disturbances in Friedreich's ataxia platelets.</a> Bioanalysis. 7 (15), 1843-1855 (2015).</li><li>Worth, A. J., Basu, S. S., Snyder, N. W., Mesaros, C., Blair, I. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inhibition+of+neuronal+cell+mitochondrial+complex+I+with+rotenone+increases+lipid+beta-oxidation,+supporting+acetyl-coenzyme+A+levels.">Inhibition of neuronal cell mitochondrial complex I with rotenone increases lipid beta-oxidation, supporting acetyl-coenzyme A levels.</a> J. Biol. Chem. 289 (39), 26895-26903 (2014).</li><li>Zhou, L., Schmaier, A. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Platelet+aggregation+testing+in+platelet-rich+plasma:+description+of+procedures+with+the+aim+to+develop+standards+in+the+field.">Platelet aggregation testing in platelet-rich plasma: description of procedures with the aim to develop standards in the field.</a> Am. J. Clin. Pathol. 123 (2), 172-183 (2005).</li></ol>

作者宣称没有竞争经济利益。

在这里，我们已经表明孤立血小板的效用作为研究扰动线粒体代谢的平台。具体地，我们已响应于鱼藤酮复合物I抑制其特征代谢适应。 目前的研究复杂我抑制细胞系对人类血小板的作用鱼藤酮扩展先前报告的调查结果。重要的是，这已表明，鱼藤酮也抑制血小板琥珀酰-CoA的形成，刺激在血小板βHB辅酶A的增加和对乙酰-CoA的血小板水平没有影响。 极大必?...

功能失调线粒体代谢已牵涉多种疾病，包括神经变性，肿瘤和心血管疾病30。这样，巨大的努力已被放在特征有助于疾病的发病机制的代谢缺陷。液相色谱-串联质谱（LC-MS / MS）被认为是用于从复杂的生物基质分析物定量的黄金标准，并经常用于代谢研究8。然而，由于经常与生物医学研究中的情况下，获得有关人类疾病可访问的和明确的模型是一个挑战。 许多研究聘请转化的细胞模型，用于探测生物异源物质或遗传异常的对细胞代谢7,9的影响。发生在肿瘤细胞中可引入混杂代谢重新编程因子21，因此不理想。这些问题可以是circumve原发性细胞模型nted，虽然获得足够的生物质的代谢分析可以是具有挑战性的。此外，大量的文化中使用抗生素的影响已经凸显为潜在的混杂线粒体研究16。 人血小板无力利用与代谢研究5,22,27,32足够的线粒体内容主要细胞模型的机会。首先，血小板可容易地获得的，通过抽血从个体供体，或从血库大量，因此提供其中外部因素可容易地控制的模型。其次，由于它们的尺寸小，血小板可以很容易地从其它血液成分以最小的准备工作中，即使最小装备的实验室5隔离。值得注意的是，血小板不含有晶核，因此可以被用来改变独立地转录调控的研究来代谢。这里，我们表明除了酰基辅酶A（辅酶A）硫酯的相对定量，分离的血小板系统可用于检查碳代谢。具体地讲，我们报道了使用代谢标记的稳定同位素（非放射性）标记的[13 C 6]葡萄糖和[13 C 16] -palmitate探测的[13℃] -label掺入重要代谢产物乙酰经由糖酵解或脂肪酸氧化辅酶A.这提供了一个有力的，概括的，和多功能平台由于酰基-CoA物种的生化途径13,24广泛参与和该系统对测试其他变量，如复合物I的抑制鱼藤酮3,33的易处理性。除了 ​​下面的协议中提供的信息，用于对同位素标记和用于基于LC-MS-分析的方法的广泛的说明可在巴苏和Blair 4中找到。

<table><tbody><tr><td>&lt;strong&gt;Reagent&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Sodium Chloride (NaCl)</td><td>Sigma-Aldrich</td><td>746398</td><td></td></tr><tr><td>Sodium Bicarbonate (NaHCO&lt;sub&gt;3&lt;/sub&gt;)</td><td>Sigma-Aldrich</td><td>S5761</td><td></td></tr><tr><td>Calcium Chloride Dihydrate (CaCl&lt;sub&gt;2&lt;/sub&gt; * H&lt;sub&gt;2&lt;/sub&gt;O)</td><td>Sigma-Aldrich</td><td>223506</td><td></td></tr><tr><td>Potassium Chloride (KCl)</td><td>Sigma-Aldrich</td><td>P9541</td><td></td></tr><tr><td>Magnesium Chloride (MgCl&lt;sub&gt;2&lt;/sub&gt;)</td><td>Sigma-Aldrich</td><td>208337</td><td></td></tr><tr><td>Glucose</td><td>Sigma-Aldrich</td><td>G8270</td><td></td></tr><tr><td>&lt;sup&gt;13&lt;/sup&gt;C&lt;sub&gt;6&lt;/sub&gt;-Glucose</td><td>Sigma-Aldrich</td><td>389374</td><td></td></tr><tr><td>Palmitic acid</td><td>Cayman</td><td>10006627</td><td></td></tr><tr><td>&lt;sup&gt;13&lt;/sup&gt;C&lt;sub&gt;16&lt;/sub&gt;-Palmitic Acid</td><td>Sigma-Aldrich</td><td>605573</td><td></td></tr><tr><td>Rotenone</td><td>Sigma-Aldrich</td><td>R8875</td><td></td></tr><tr><td>Trichloro Acetic Acid</td><td>Sigma-Aldrich</td><td>T6399</td><td></td></tr><tr><td>5-Sulfosalicylic Acid</td><td>Sigma-Aldrich</td><td>390275</td><td></td></tr><tr><td>Acetonitirle</td><td>Fischer Scientific</td><td>A996-4</td><td>(optima)</td></tr><tr><td>Water (H&lt;sub&gt;2&lt;/sub&gt;O)</td><td>Fischer Scientific</td><td>W7-4</td><td>(optima)</td></tr><tr><td>Formic acid</td><td>Fischer Scientific</td><td>85171</td><td>(optima)</td></tr><tr><td>Dimethyl Sulfoxide</td><td>Sigma-Aldrich</td><td>472301</td><td></td></tr><tr><td>Ethanol</td><td>Fischer Scientific</td><td>04-355-222</td><td></td></tr><tr><td>Methanol</td><td>Fischer Scientific</td><td>A454-4</td><td>(optima)</td></tr><tr><td>Ammonium Acetate</td><td>Fischer Scientific</td><td>A639-500</td><td></td></tr><tr><td>2 ml Eppendorf Tubes</td><td>BioExpress</td><td>C-3229-1</td><td></td></tr><tr><td>LC vials (plastic)</td><td>Waters</td><td>186002640</td><td></td></tr><tr><td>10 ml Glass Centrifuge Tubes</td><td>Kimble Chase</td><td>73785-10</td><td></td></tr><tr><td>Oasis Solid Phase Extraxtion (SPE) Columns</td><td>Waters</td><td>WAT094225</td><td></td></tr><tr><td>Pastuer Pipets</td><td>Fischer Scientific</td><td>13-678-200</td><td></td></tr><tr><td>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;Equipment&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>CO&lt;sub&gt;2&lt;/sub&gt; Water-Jacketed Incubator</td><td>Nuaire</td><td>AutoFlow NU-8500</td><td></td></tr><tr><td>Triple Quadropole Mass Spectrometer</td><td>Thermo Scientific</td><td>Finnigan TSQ Quantum</td><td></td></tr><tr><td>HPLC</td><td>Thermo Scientific</td><td>Dionex Ultimate 3000</td><td></td></tr><tr><td>Source</td><td>Thermo Scientific</td><td>HESI II</td><td></td></tr><tr><td>HPLC Column</td><td>Phenomenex</td><td>Luna C18</td><td>3 &amp;mu;m particle size, 200 mm x 2 mm</td></tr></tbody></table>

lc-ms analysis of human platelets as a platform for studying mitochondrial metabolism

Perturbed mitochondrial metabolism has received renewed interest as playing a causative role in a range of diseases. Probing alterations to metabolic pathways requires a model in which external factors can be well controlled, allowing for reproducible and meaningful results. Many studies employ transformed cellular models for these purposes; however, metabolic reprogramming that occurs in many cancer cell lines may introduce confounding variables. For this reason primary cells are desirable, though attaining adequate biomass for metabolic studies can be challenging. Here we show that human platelets can be utilized as a platform to carry out metabolic studies in combination with liquid chromatography-tandem mass spectrometry analysis. This approach is amenable to relative quantification and isotopic labeling to probe the activity of specific metabolic pathways. Availability of platelets from individual donors or from blood banks makes this model system applicable to clinical studies and feasible to scale up. Here we utilize isolated platelets to confirm previously identified compensatory metabolic shifts in response to the complex I inhibitor rotenone. More specifically, a decrease in glycolysis is accompanied by an increase in fatty acid oxidation to maintain acetyl-CoA levels. Our results show that platelets can be used as an easily accessible and medically relevant model to probe the effects of xenobiotics on cellular metabolism.

为了证明这种方法我们已经再现先前描述代偿代谢适应的所得暴露于鱼藤酮概的效用。这一发现在细胞培养模型先前确定和本调查的目的，可测试此代谢移也发生在血小板，是无核，不容易在相同实验工件作为细胞培养物。这项工作是用6天的血小板宾夕法尼亚创伤中心，已被认为太老了人类输液进行的，虽然他们保留了他们的代谢活动。 如图1中进行了隔离?...

Watch this Scientific Journal Video about LC-MS Analysis of Human Platelets as a Platform for Studying Mitochondrial Metabolism at JoVE.com

LC-MS Analysis of Human Platelets as a Platform for Studying Mitochondrial Metabolism

这里，我们表明分离的人血小板可以用作一个可访问的体外模型来研究的代谢适应响应于I抑制剂鱼藤酮的复合物。这种方法通过液相色谱 - 质谱法使用同位素示踪和相对定量，并且可以应用于各种研究设计的。

人类血小板的LC-MS分析作为研究线粒体代谢的平台

Perturbed mitochondrial metabolism has received renewed interest as playing a causative role in a range of diseases. Probing alterations to metabolic ...

lc-ms-analysis-human-platelets-as-platform-for-studying-mitochondrial

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11.1K 次观看.  University of Pennsylvania. 这里，我们表明分离的人血小板可以用作一个可访问的体外模型来研究的代谢适应响应于I抑制剂鱼藤酮的复合物。这种方法通过液相色谱 - 质谱法使用同位素示踪和相对定量，并且可以应用于各种研究设计的。 

视频: LC-MS Analysis of Human Platelets as a Platform for Studying Mitochondrial Metabolism

Analyses of Mitochondrial Calcium Influx in Isolated Mitochondria and Cultured Cells

Ca2+ handling by mitochondria is a critical function regulating both physiological and pathophysiological processes in a broad spectrum of cells. The ability to accurately measure the influx and efflux of Ca2+ from mitochondria is important for determining the role of mitochondrial Ca2+ handling in these processes. In this report, we present two methods for the measurement of mitochondrial Ca2+ handling in both isolated mitochondria and cultured cells. We first detail a plate reader-based platform for measuring mitochondrial Ca2+ uptake using the Ca2+ sensitive dye calcium green-5N. The plate reader-based format circumvents the need for specialized equipment, and the calcium green-5N dye is ideally suited for measuring Ca2+ from isolated tissue mitochondria. For our application, we describe the measurement of mitochondrial Ca2+ uptake in mitochondria isolated from mouse heart tissue; however, this procedure can be applied to measure mitochondrial Ca2+ uptake in mitochondria isolated from other tissues such as liver, skeletal muscle, and brain. Secondly, we describe a confocal microscopy-based assay for measurement of mitochondrial Ca2+ in permeabilized cells using the Ca2+ sensitive dye Rhod-2/AM and imaging using 2-dimensional laser-scanning microscopy. This permeabilization protocol eliminates cytosolic dye contamination, allowing for specific recording of changes in mitochondrial Ca2+. Moreover, laser-scanning microscopy allows for high frame rates to capture rapid changes in mitochondrial Ca2+ in response to various drugs or reagents applied in the external solution. This protocol can be applied to measure mitochondrial Ca2+ uptake in many cell types including primary cells such as cardiac myocytes and neurons, and immortalized cell lines.

Here, we present two protocols for the measurement of mitochondrial Ca2+ influx in isolated mitochondria and cultured cells. For isolated mitochondria, we detail a plate reader-based Ca2+ import assay using the Ca2+ sensitive dye calcium green-5N. For cultured cells, we describe a confocal microscopy method using the Ca2+ dye Rhod-2/AM.

线粒体钙在分离线粒体和培养细胞中的流入分析

Ca2+ handling by mitochondria is a critical function regulating both physiological and pathophysiological processes in a broad spectrum of cells. The ...

科研

生物化学

Flow Cytometric Analysis of Mitochondrial Reactive Oxygen Species in Murine Hematopoietic Stem and Progenitor Cells and MLL-AF9 Driven Leukemia

We present a flow cytometric approach for analyzing mitochondrial ROS in various live bone marrow (BM)-derived stem and progenitor cell populations from healthy mice as well as mice with AML driven by MLL-AF9. Specifically, we describe a two-step cell staining process, whereby healthy or leukemia BM cells are first stained with a fluorogenic dye that detects mitochondrial superoxides, followed by staining with fluorochrome-linked monoclonal antibodies that are used to distinguish various healthy and malignant hematopoietic progenitor populations. We also provide a strategy for acquiring and analyzing the samples by flow cytometry. The entire protocol can be carried out in a timeframe as short as 3-4 h. We also highlight the key variables to consider as well as the advantages and limitations of monitoring ROS production in the mitochondrial compartment of live hematopoietic and leukemia stem and progenitor subpopulations using fluorogenic dyes by flow cytometry. Furthermore, we present data that mitochondrial ROS abundance varies among distinct healthy HSPC sub-populations and leukemia progenitors and discuss the possible applications of this technique in hematologic research.

We describe a method for using multiparameter flow cytometry to detect mitochondrial reactive oxygen species (ROS) in murine healthy hematopoietic stem and progenitor cells (HSPCs) and leukemia cells from a mouse model of acute myeloid leukemia (AML) driven by MLL-AF9.

木尿造血干细胞和祖细胞和MLL-AF9驱动白血病线粒体反应氧物种的流细胞学分析

We present a flow cytometric approach for analyzing mitochondrial ROS in various live bone marrow (BM)-derived stem and progenitor cell populations from ...

癌症研究

Bioenergetics and the Oxidative Burst: Protocols for the Isolation and Evaluation of Human Leukocytes and Platelets

Mitochondrial dysfunction is known to play a significant role in a number of pathological conditions such as atherosclerosis, diabetes, septic shock, and neurodegenerative diseases but assessing changes in bioenergetic function in patients is challenging.&#160;Although diseases such as diabetes or atherosclerosis present clinically with specific organ impairment, the systemic components of the pathology, such as hyperglycemia or inflammation, can alter bioenergetic function in circulating leukocytes or platelets. This concept has been recognized for some time but its widespread application has been constrained by the large number of primary cells needed for bioenergetic analysis.&#160;This technical limitation has been overcome by combining the specificity of the magnetic bead isolation techniques, cell adhesion techniques, which allow cells to be attached without activation to microplates, and the sensitivity of new technologies designed for high throughput microplate respirometry.&#160;An example of this equipment is the extracellular flux analyzer. Such instrumentation typically uses oxygen and pH sensitive probes to measure rates of change in these parameters in adherent cells, which can then be related to metabolism. Here we detail the methods for the isolation and plating of monocytes, lymphocytes, neutrophils and platelets, without activation, from human blood and the analysis of mitochondrial bioenergetic function in these cells. In addition, we demonstrate how the oxidative burst in monocytes and neutrophils can also be measured in the same samples. Since these methods use only 8-20 ml human blood they have potential for monitoring reactive oxygen species generation and bioenergetics in a clinical setting.

Blood leukocytes and platelets can be used as a marker of overall bioenergetic health of an individual and so have the potential to monitor pathological processes and the impact of treatments. Here&#160;we describe a method to isolate and measure mitochondrial function and the oxidative burst in these cells.

生物能量和氧化突发：协议用于人类白细胞和血小板的隔离与评价

Mitochondrial dysfunction is known to play a significant role in a number of pathological conditions such as atherosclerosis, diabetes, septic shock, and ...

免疫与感染

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 ...

Flow Cytometric Analysis of Multiple Mitochondrial Parameters in Human Induced Pluripotent Stem Cells and Their Neural and Glial Derivatives

Mitochondria are important in the pathophysiology of many neurodegenerative diseases. Changes in mitochondrial volume, mitochondrial membrane potential (MMP), mitochondrial production of reactive oxygen species (ROS), and mitochondrial DNA (mtDNA) copy number are often features of these processes. This report details a novel flow cytometry-based approach to measure multiple mitochondrial parameters in different cell types, including human induced pluripotent stem cells (iPSCs) and iPSC-derived neural and glial cells. This flow-based strategy uses live cells to measure mitochondrial volume, MMP, and ROS levels, as well as fixed cells to estimate components of the mitochondrial respiratory chain (MRC) and mtDNA-associated proteins such as mitochondrial transcription factor A (TFAM). 
By co-staining with fluorescent reporters, including MitoTracker Green (MTG), tetramethylrhodamine ethyl ester (TMRE), and MitoSox Red, changes in mitochondrial volume, MMP, and mitochondrial ROS can be quantified and related to mitochondrial content. Double staining with antibodies against MRC complex subunits and translocase of outer mitochondrial membrane 20 (TOMM20) permits the assessment of MRC subunit expression. As the amount of TFAM is proportional to mtDNA copy number, the measurement of TFAM per TOMM20 gives an indirect measurement of mtDNA per mitochondrial volume. The entire protocol can be carried out within 2-3 h. Importantly, these protocols allow the measurement of mitochondrial parameters, both at the total level and the specific level per mitochondrial volume, using flow cytometry.

This study reports a novel approach to measure multiple mitochondrial functional parameters based on flow cytometry and double staining with two fluorescent reporters or antibodies to detect changes in mitochondrial volume, mitochondrial membrane potential, reactive oxygen species level, mitochondrial respiratory chain composition, and mitochondrial DNA.

人诱导多能干细胞及其神经和神经胶质衍生物中多种线粒体参数的流式细胞术分析

Mitochondria are important in the pathophysiology of many neurodegenerative diseases. Changes in mitochondrial volume, mitochondrial membrane potential ...

神经科学

Analysis of Mitochondrial Respiratory Chain Complexes in Cultured Human Cells using Blue Native Polyacrylamide Gel Electrophoresis and Immunoblotting

Mitochondrial respiration is performed by oxidative phosphorylation (OXPHOS) complexes within mitochondria. Internal and environmental factors can perturb the assembly and stability of OXPHOS complexes. This protocol describes the analysis of mitochondrial respiratory chain complexes by blue native polyacrylamide gel electrophoresis (BN-PAGE) in application to cultured human cells. First, mitochondria are extracted from the cells using digitonin, then using lauryl maltoside, the intact OXPHOS complexes are isolated from the mitochondrial membranes. The OXPHOS complexes are then resolved by gradient gel electrophoresis in the presence of the negatively charged dye, Coomassie blue, which prevents protein aggregation and ensures electrophoretic mobility of protein complexes towards the cathode. Finally, the OXPHOS complexes are detected by standard immunoblotting. Thus, BN-PAGE is a convenient and inexpensive technique that can be used to evaluate the assembly of entire OXPHOS complexes, in contrast to the basic SDS-PAGE allowing the study of only individual OXPHOS complex subunits.

This protocol demonstrates the analysis of mitochondrial respiratory chain complexes by blue native polyacrylamide gel electrophoresis. Here the method applied to cultured human cells is described.

蓝色原代聚丙烯酰胺凝胶电泳免疫印迹法诱导人细胞线粒体呼吸链配合物的分析

Mitochondrial respiration is performed by oxidative phosphorylation (OXPHOS) complexes within mitochondria. Internal and environmental factors can perturb ...

生物学

人 PBMC 中线粒体功能的高分辨率分析

High-Resolution Fluorespirometry to Assess Dynamic Changes in Mitochondrial Membrane Potential in Human Immune Cells

Peripheral mononuclear cells (PBMCs) exhibit robust changes in mitochondrial respiratory capacity in response to health and disease. While these changes&#160;do not always reflect what occurs in other tissues, such as skeletal muscle, these cells are an accessible and valuable source of viable mitochondria from human subjects. PBMCs are exposed to systemic signals that impact their bioenergetic state. Thus, expanding our tools to interrogate mitochondrial metabolism in this population will elucidate mechanisms related to disease progression. Functional assays of mitochondria are often limited to using respiratory outputs following maximal substrate, inhibitor, and uncoupler concentrations to determine the full range of respiratory capacity, which may not be achievable in vivo. The conversion of adenosine diphosphate (ADP) to adenosine triphosphate (ATP) by ATP-synthase results in a decrease in mitochondrial membrane potential (mMP) and an increase in oxygen consumption. To provide a more integrated analysis of mitochondrial dynamics, this article describes the use of high-resolution fluorespirometry to measure the simultaneous response of oxygen consumption and mitochondrial membrane potential (mMP) to physiologically relevant concentrations of ADP. This technique uses tetramethylrhodamine methylester (TMRM) to measure mMP polarization in response to ADP titrations following maximal hyperpolarization with complex I and II substrates. This technique can be used to quantify how changes in health status, such as aging and metabolic disease, affect the sensitivity of mitochondrial response to energy demand in PBMCs, T-cells, and monocytes from human subjects.

作者聚焦：使用荧光螺线测定法对 PBMC 线粒体反应的新见解

Methods for studying mitochondrial bioenergetics under physiologically relevant substrate concentrations in immune cells are limited. We provide a detailed protocol that uses high-resolution fluorespirometry to assess changes in the response of the mitochondrial membrane potential to energy demand in human T-cells, monocytes, and peripheral mononuclear cells.

人类血小板的LC-MS分析作为研究线粒体代谢的平台

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