这项工作由加拿大自然科学和工程研究委员会 (NSERC) 资助。视频制作是与纽芬兰纪念大学教与学创新中心 (CITL) 合作进行的。

1. 化学物质和纯化酶
<ol><li>采购以下材料: 猪心源的 PDHC 和 KGDHC (或另一种纯化的线粒体 flavoenzyme);H2O2 (30% 解决方案), 丙酮酸、αα-、NAD+、NADH、CoASH、磷酸硫胺 (TPP)、甘露醇、HEPES、蔗糖、EGTA、3-甲基2羰基戊酸 (KMV)、超氧化物歧化酶 (SOD)、辣根过氧化物酶 (HRP)、10-乙酰基 37-dihydroxyphenoxazine 试剂 (AUR) 和 CPI-613。</li>
</ol>2. 计划化验及试剂制备
<ol><li>根据96井板中的表 1设置检测。 注: 每种反应的总容积为200µL. 调整库存浓度, 使20µL 试剂添加到每个井中。这确保了在开始试验之前, 快速添加辅助因子和基底对每个反应良好。</li>
	<li>制备含有220毫米甘露醇、70毫米蔗糖、1毫米 EGTA 和20毫米 HEPES (pH 7.4 与 HCl) 的反应缓冲液。 注意: 这可能会根据不同的纯化酶的物理特性而改变。</li>
	<li>使用酶活性测定法检查纯化的 KGDHC 或 PDHC 的污染情况14,16,17或应用免疫印迹或考马斯着色5。</li>
	<li>根据表 1中的工作溶液浓度, 准备反应缓冲器中的所有试剂。</li>
	<li>将所有试剂贮存为1毫升整除数在-20 摄氏度。在 100-µL 整除数中储存丙酮酸和αα-, 以防止自氧化和反复冻融导致的自发降解。</li>
	<li>在1毫升亚砜中制备 AUR 试剂, 然后在反应缓冲液中稀释至100µM。存储保护不受光照。 注: 在这里, 100 µM 工作解决方案是新鲜的每2到3周, 并保护免受光。</li>
</ol>3. 建立化验模板
<ol><li>在具有双测量能力的单色微板块阅读器上设置检测程序。</li>
	<li>通过选择 "协议" 来定义阅读过程。然后单击 "过程"。</li>
	<li>将 "温度" 设置为25°c (图 1A)。</li>
	<li>单击 "启动动能" 以设置读取条件 (图 1A)。设置读取间隔/实验的时间和数量。</li>
	<li>单击 "读取" (图 1B)。 注: 样品从顶部的位置读取, 增益为50。时间: 5 分钟, 以30秒为间隔读取。通道 1: 试卤灵荧光激发/发射 = 565 nm:600 nm, 波长宽度 13.5 nm。通道 2: NADH 自发荧光-激发/发射 = 376 nm:450 nm, 波长宽度 20 nm。</li>
	<li>在 "读取" 下, 选择要监视的水井 (例如、A1-A4 和 B1-B4)。</li>
	<li>选择 "结束动能"。</li>
</ol>4. 标准曲线
<ol><li>解冻试剂和储存在冰, 直到需要。</li>
	<li>
NADH 标准曲线 (图 2A)
	<ol>
<li>在反应缓冲液中, 为 0.5-10 毫米的浓度范围准备 NADH 的工作溶液。</li>
		<li>将每个 NADH 工作溶液浓度的20µL 整除数添加到含有180µL 反应缓冲器的井中。NADH 在每个井中的最终浓度为0.05-1 毫米。</li>
		<li>单击 "读取" 并设置激发/发射 = 376 nm:450 nm, 波长宽度为 20 nm。 注意: 这是只读端点-不需要动能参数。</li>
	</ol>
</li>
	<li>
AUR 标准曲线 (图 2B)
	<ol>
<li>在反应缓冲器中制备过氧化氢的工作储存;库存浓度从 200-4, 000 nM 不等。</li>
		<li>将反应缓冲器的120µL 添加到每个井中。</li>
		<li>添加20µL (工作库存浓度 = 30 U/毫升, 最后集中在井 = 3 U/毫升), 20 µL SOD (工作存货集中 = 250 u/毫升, 最后集中在井 = 25 U/毫升) 和20µL AUR (工作存货集中 = 100 µM, 最后集中在井 = 10 µM) 对每个井包含反应缓冲。</li>
		<li>将每个 H2O2工作库存解决方案的20µL 添加到每个包含缓冲区和检测试剂的井中。最终反应量为200µL, 每个井中 H2O2的最终浓度为 20-400 nM。</li>
		<li>在25摄氏度的平板阅读器中孵化出1分钟的板材。</li>
		<li>单击 "读取" 并设置激发/发射 = 565 nm/600 nm, 波长宽度为 13.5 nm。请注意, 这是只读端点-不需要动能参数。</li>
	</ol>
</li>
</ol>5. 测量 O2/H 2 O2发布和 NADH 生产由 KGDHC 和 PDHC
<ol><li>有关不同试剂的添加顺序、每个工作解决方案的浓度以及每个井中添加的卷, 请参见表 1 。</li>
	<li>每井加40µL 反应缓冲液。对于使用 KMV 或 CPI-613 的检测, 请在每个井中添加20µL 缓冲区。</li>
	<li>添加20µL 的 PDHC 或 KGDHC (工作库存的 1 U/毫升, 最终浓度每井 = 0.1 U/毫升) 每个井。</li>
	<li>孵育 PDHC 和 KGDHC 在25°c 2 分钟。</li>
	<li>添加20µL 的 KMV (工作库存 = 100 毫米, 最终浓度 = 10 毫米) 或20µL CPI-613 (工作库存浓度 = 1.5 毫米, 最终浓度 = 150 µM) (表 2)。 注: 如果抑制剂未用于化验, 则可排除此步骤。</li>
	<li>孵育1分钟, 在25摄氏度。 注: 如果抑制剂未用于化验, 则可排除此步骤。</li>
	<li>添加20µL (工作库存浓度 = 30 单位/毫升, 最终浓度在井 = 3 单位/毫升) 和20µL 的 SOD (工作库存浓度 = 250 单位/毫升, 最终集中在井 = 25 单位/毫升) 对每个井。</li>
	<li>添加20µL 辅酶 A (工作库存浓度 = 1 毫米, 最后集中 = 0.1 毫米), TPP 的20µL (工作存货集中 = 3 毫米, 最后集中 = 0.3 毫米) 和20µL 的 NAD+ (工作存货集中 = 10 毫米, 最后集中 = 1 毫米) 对每个 w告诉.</li>
	<li>将 AUR 试剂从保护性锡纸覆盖物中取出, 并在每个井中加20µL。</li>
	<li>添加20µL 丙酮酸盐 (工作存货浓度从1-100 毫米, 最后浓度为化验 = 0.1-10 毫米) 到含 PDHC 和αα-的水井 (工作库存浓度不等于1-100 毫米, 最终浓度为化验 = 0.1-10 毫米) 到含 KGDHC 的水井。</li>
	<li>单击 "读取" 开始动态测量。</li>
</ol>6. 数据分析
<ol><li>按住键盘上的 "CTRL" 按钮, 然后单击 "水井" 生成一个包含与1通道 (图 3A) 对应的所有动态测量的图表。</li>
	<li>在不同时间点 (图 3B) 测量的相对荧光单元 (RFU) 的原始值, 单击右上角的视图图标中的 "数据"。</li>
	<li>导出分析值 (表 3)。</li>
	<li>单击右上方的 "图表" 下拉菜单 (图 3B) 可访问与荧光通道2关联的 RFU 值。</li>
	<li>对通道2重复步骤6.1 到6.3。</li>
</ol>

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没有什么可透露的。

该协议是有利的, 因为, 1) 它消除了任何可能干扰 H2O2检测 (如, 抗氧化剂系统或其他 ROS 来源), 2) 的相互竞争的反应, 提供直接评估的本地率ros 释放的黄素线粒体脱氢酶, 3) 允许比较的本地 ros 释放率的两个或更多的纯化黄素脱氢酶, 4) 可以允许直接比较 ros 释放率和酶活性, 和 5)允许筛选活性氧释放的选择性竞争抑制剂。我们的小组和其他人在以前的出版物中使用这种方法来?...

营养代谢的最终目的是使三磷酸腺苷 (ATP)。在哺乳动物细胞中, 这发生在线粒体, 双 membraned 细胞器, 将储存在碳中的能量转化为 ATP。ATP 的产生是在碳被形成两个电子载体、NADH 和黄素腺嘌呤二核苷酸 (FADH2)1的线粒体燃烧时开始的。NADH 和 FADH2分别由多亚基呼吸复合体 I 和 II 氧化, 并且被解放的电子被运到终端电子受体分子氧 (O2) 在复杂 IV1。热力学上有利的 "下坡" 转移电子到O 2 在链子的末端被结合对质子的出口入膜间隙空间由复合体 I, III 和 iv。这创造了质子的跨膜电化学梯度 (质子动力), 一种临时形式的吉布斯自由能, 被复杂的 V 挖掘, 使 ATP2。线粒体中的电子转移反应与 ATP 生产不完全耦合。在克雷布斯周期和呼吸链的不同点, 电子可以过早地与 O2相互作用, 形成 ROS3。线粒体产生的最生物相关的 ROS 为 O2和 H 2O2。尽管 O2通常被认为是由线粒体形成的近端 ROS, 但现在明显的是, 生产地点形成了 O2和 H 2O2的混合物, 这与自由黄素假肢组的激进化学4,5。在高水平, ROS 可能是危险的, 破坏细胞功能所需的生物成分, 导致氧化窘迫6。然而, 在低数量, 线粒体 ROS 实现重要的信号功能。例如, 从线粒体释放的 H2O2被牵连到控制 T 细胞活化、应力信号 (例如、诱导 Nrf2 信号通路)、诱导细胞增殖和分化,胰岛素信号和释放, 和喂养行为7。在了解 ROS 信号功能方面取得了相当大的进展。然而, 对于线粒体中的酶作为最重要的来源和如何控制生产, 仍然存在重要的问题。
ROS 的释放由一个地点生产取决于几个因素: 1) 电子捐赠站点的集中, 2) 电子捐赠站点的氧化还原状态, 3) 获得氧气, 和 4) 氧化基底的浓度和类型3,8,9. 在线粒体中, 其他因素, 如 NADH 的浓度和膜电位强度也影响 ROS 生产8,10。例如, O2/H2O2生产的速率由纯化的 PDHC 或 KGDHC 增加, NADH 可用性5,11。在这种情况下, 电子从 NADH 向后流向 PDHC 或 KGDHC E3亚基中的时尚中心, O2/H2O2生产12的站点。同样, 提供的 NAD+具有相反的效果, 减少了 KGDHC12中的 ROS 释放。因此, 控制来自 ROS 生产场所的电子进入或退出可以改变多少 O2/H2O2的形成。例如, 用 CPI-613 (硫辛酸模拟) 阻止 PDHC 或 KGDHC 的 E2亚基, 在 O2/H2O2生产13中, 结果几乎减少90%。类似的结果可以获得与化学 s-glutathionylation 催化剂, 胍和双硫醒, 几乎废除O2/H2O 2 生产由 PDHC 或 KGDHC 通过共轭谷胱甘肽到 E2亚基14。通过 PDHC 和 KGDHC E2亚基阻断电子流的权衡是 NADH 产量的减少, 这降低了电子传输链 (例如, 复杂 III) 的 ROS 形成。这也可以减少电子传输链的 ATP 输出。总的来说, 阻止电子进入 ROS 生产场所可以是控制 O2/H2o2生产的高效方法。
线粒体最多可包含 12个潜在 O2/H2O2源6。这些站点中的大多数都是黄素的酶, 它们生成了 O2和 H 2O2的混合物。使用不同的基质和抑制剂组合, 可以确定哪些呼吸复合体和线粒体脱氢酶作为高容量 O 2/H 2 O 2 形成站点在不同的组织3。PDHC 和 KGDHC 已被证明为高容量 O2/H2O2在肌肉和肝脏线粒体13,15发射站点。但是, 在检查 O2/H2O2形成线粒体中单个站点的潜力以及不同基质和抑制剂组合对其产生的影响方面仍存在一些困难酶的活性。这是由于存在不需要的副作用 (例如, 形成 O2/H2O2由非兴趣酵素以外的站点), 污染内源营养素 (例如,脂肪酸) 或细胞器(例如,, 过氧化物酶体也形成O2-/H2O2), 使用缺乏选择性的抑制剂和/或使用不完全抑制 ROS 生产的化合物。某些抑制剂还能改变线粒体的生物能学和电子流的方向, 这就改变了 ROS 从其他生产场所释放出来的活性和混淆结果。o2/H2O2 从单个站点释放的绝对速率也难以量化由于 O2-和 H2o2消除基质中的酶和膜间隙空间。因此, 消除可能干扰 o2/H2O2发布测量的任何相互竞争的反应, 在识别高容量 O2/2o2 时, 都很有用。形成站点。
在这里, 我们提出一个简单的方法, 允许同时检查 O2/H2O2生产和 NADH 形成由纯化的黄素依赖脱氢酶。通过使用纯化的酶, 不需要的 ros 形成的副作用和 ros 降解酶可以消除, 允许更准确地测量本地 O2/H2o2生产率为个别 flavoenzymes。此方法可用于直接比较 o2/H2O2的不同纯化脱氢酶的形成能力, 或为 o 2-/h 2 o 筛选潜在的站点特定抑制剂。2版本。最后, 测量 O2/H2O2和 NADH 生产同时可以对酶活性与 ROS 释放能力之间的关系进行实时评估。

<table><tbody><tr><td>Pyruvate dehydrogenase complex</td><td>SIGMA</td><td>P7032-10UN</td><td>purified flavoenzyme</td></tr><tr><td>alpha-ketoglutarate dehydrogenase complex</td><td>SIGMA</td><td>K1502-20UN</td><td>purified flavoenzyme</td></tr><tr><td>30% hydrogen peroxide solution</td><td>SIGMA</td><td>HX0640-5</td><td>reagent, standard curves</td></tr><tr><td>NAD+</td><td>SIGMA</td><td>N0632-1G</td><td>reagent, activity/ROS release assay</td></tr><tr><td>NADH</td><td>SIGMA</td><td>N4505-100MG</td><td>reagent, standard curves</td></tr><tr><td>pyruvate</td><td>SIGMA</td><td>P2256-5G</td><td>reagent, activity/ROS release assay</td></tr><tr><td>alpha-ketoglutarate</td><td>SIGMA</td><td>75892-25G</td><td>reagent, activity/ROS release assay</td></tr><tr><td>CoASH</td><td>SIGMA</td><td>C3019-25MG</td><td>reagent, activity/ROS release assay</td></tr><tr><td>thiamine pyrophosphate</td><td>SIGMA</td><td>C8754-1G</td><td>reagent, activity/ROS release assay</td></tr><tr><td>mannitol</td><td>SIGMA</td><td>M4125-100G</td><td>buffer component</td></tr><tr><td>Hepes</td><td>SIGMA</td><td>H3375-25G</td><td>buffer component</td></tr><tr><td>sucrose</td><td>SIGMA</td><td>S7903-250G</td><td>buffer component</td></tr><tr><td>EGTA</td><td>SIGMA</td><td>E3889-10G</td><td>buffer component</td></tr><tr><td>KMV</td><td>SIGMA</td><td>198978-5G</td><td>reagent, ROS release inhibitor</td></tr><tr><td>CPI-613</td><td>Santa Cruz</td><td>sc-482709</td><td>reagent, ROS release inhibitor</td></tr><tr><td>SOD</td><td>SIGMA</td><td>S9697-15KU</td><td>reagent, ROS release detection</td></tr><tr><td>horseradish peroxidase</td><td>SIGMA</td><td>P8375-1KU</td><td>reagent, ROS release detection</td></tr><tr><td>Amplex Ultra Red</td><td>Thermofisher</td><td>A36006</td><td>reagent, ROS release detection</td></tr><tr><td>Biotech Synergy 2 microplate reader</td><td>BioTek Instruments</td><td></td><td>microplate reader for assays</td></tr><tr><td>Gen5 software</td><td>BioTek Instruments</td><td></td><td>software, used for collection of raw RFU</td></tr><tr><td>Graphpad Prism</td><td>Graphpad software</td><td></td><td>software, data analysis</td></tr><tr><td>Microsoft EXCEL</td><td>Microsoft</td><td></td><td>software, data analysis</td></tr></tbody></table>

simultaneous measurement of superoxide/hydrogen peroxide and nadh production by flavin-containing mitochondrial dehydrogenases

据报道, 线粒体可以包含多达12种酶源的活性氧 (ROS)。大多数这些网站包括黄素依赖的呼吸复合体和脱氢酶, 产生超氧化物的混合物 (O 2) 和过氧化氢 (H 2 O 2).由于抗氧化剂防御系统的存在和侧面反应, 也形成 O2/H2O2, 因此, 对孤立线粒体中单个站点的 ROS 产生潜能的准确量化可能会有挑战性。使用可破坏线粒体生物能学的非特异性抑制剂也可以通过改变其他生产场所的 ROS 释放来降低测量结果。在这里, 我们提出了一个简单的方法, 同时测量 H2O2释放和烟酰胺腺嘌呤二核苷酸 (NADH) 生产的纯化黄素链脱氢酶。为了我们的目的, 我们用纯化的丙酮酸脱氢酶复合物 (PDHC) 和αα-脱氢酶复合物 (KGDHC) 的猪心源为例。此方法允许通过消除其他潜在的 ROS 和抗氧化剂系统的来源, 精确测量本机 H2O2的发布速率。此外, 该方法还可以直接比较 H2O2释放与酶活性之间的关系, 以及筛选活性抑制剂对 ROS 生产的有效性和选择性。总的来说, 这种方法可以在进行更复杂的实验时, 对单独的线粒体或透肌纤维进行更精密的试验, 从而对个体酶的原生氧释放率进行深入评估。

图 3A为在同时测量 H2O2和 NADH 生产过程中收集的 RFU 提供了一个具有代表性的跟踪。图 3B中描述了每个时间间隔的原始 RFU 数据。然后导出原始 RFU 数据以进行分析。通过从图 2中显示的标准曲线推断, 可以确定在 5 min 期间的每个测量间隔内形成的 NADH 和 H2O2的绝对量。在<stro...

Watch this Scientific Journal Video about Simultaneous Measurement of Superoxide/Hydrogen Peroxide and NADH Production by Flavin-containing Mitochondrial Dehydrogenases at JoVE.com

Simultaneous Measurement of Superoxide/Hydrogen Peroxide and NADH Production by Flavin-containing Mitochondrial Dehydrogenases

线粒体含有几种黄素依赖的酶, 可以产生活性氧 (ROS)。由于不需要的副作用, 监测线粒体中单个部位的 ROS 释放具有挑战性。我们提出了一种简单, 廉价的方法直接评估的本地率的 ROS 释放使用纯化 flavoenzymes 和微板块荧光。

含黄素线粒体脱氢酶同时测定超氧化物/过氧化氢和 NADH 生产

It has been reported that mitochondria can contain up to 12 enzymatic sources of reactive oxygen species (ROS). A majority of these sites include ...

simultaneous-measurement-superoxidehydrogen-peroxide-nadh-production

Research

JoVE Journal

Biochemistry

10.0K 次观看.  Memorial University of Newfoundland. 线粒体含有几种黄素依赖的酶, 可以产生活性氧 (ROS)。由于不需要的副作用, 监测线粒体中单个部位的 ROS 释放具有挑战性。我们提出了一种简单, 廉价的方法直接评估的本地率的 ROS 释放使用纯化 flavoenzymes 和微板块荧光。

视频: Simultaneous Measurement of Superoxide/Hydrogen Peroxide and NADH Production by Flavin-containing Mitochondrial Dehydrogenases

Monitoring the Reductive and Oxidative Half-Reactions of a Flavin-Dependent Monooxygenase using Stopped-Flow Spectrophotometry

Aspergillus fumigatus siderophore A (SidA) is an FAD-containing monooxygenase that catalyzes the hydroxylation of ornithine in the biosynthesis of hydroxamate siderophores that are essential for virulence (e.g. ferricrocin or N',N",N'''-triacetylfusarinine C)1. The reaction catalyzed by SidA can be divided into reductive and oxidative half-reactions (Scheme 1). In the reductive half-reaction, the oxidized FAD bound to Af SidA, is reduced by NADPH2,3. In the oxidative half-reaction, the reduced cofactor reacts with molecular oxygen to form a C4a-hydroperoxyflavin intermediate, which transfers an oxygen atom to ornithine. Here, we describe a procedure to measure the rates and detect the different spectral forms of SidA using a stopped-flow instrument installed in an anaerobic glove box. In the stopped-flow instrument, small volumes of reactants are rapidly mixed, and after the flow is stopped by the stop syringe (Figure 1), the spectral changes of the solution placed in the observation cell are recorded over time. In the first part of the experiment, we show how we can use the stopped-flow instrument in single mode, where the anaerobic reduction of the flavin in Af SidA by NADPH is directly measured. We then use double mixing settings where Af SidA is first anaerobically reduced by NADPH for a designated period of time in an aging loop, and then reacted with molecular oxygen in the observation cell (Figure 1). In order to perform this experiment, anaerobic buffers are necessary because when only the reductive half-reaction is monitored, any oxygen in the solutions will react with the reduced flavin cofactor and form a C4a-hydroperoxyflavin intermediate that will ultimately decay back into the oxidized flavin. This would not allow the user to accurately measure rates of reduction since there would be complete turnover of the enzyme. When the oxidative half-reaction is being studied the enzyme must be reduced in the absence of oxygen so that just the steps between reduction and oxidation are observed. One of the buffers used in this experiment is oxygen saturated so that we can study the oxidative half-reaction at higher concentrations of oxygen. These are often the procedures carried out when studying either the reductive or oxidative half-reactions with flavin-containing monooxygenases. The time scale of the pre-steady-state experiments performed with the stopped-flow is milliseconds to seconds, which allow the determination of intrinsic rate constants and the detection and identification of intermediates in the reaction4. The procedures described here can be applied to other flavin-dependent monooxygenases.5,6

We describe the use of a stopped-flow instrument to investigate both the reductive and oxidative half-reactions of Aspergillus fumigatus siderophore A (SidA), a flavin-dependent monooxygenase. We then show the spectra corresponding to the species in the reaction of SidA and we calculate the rate constants for their formation.

监视依赖黄素单加氧酶和氧化还原半反应，用停流分光光度法

Aspergillus fumigatus siderophore A (SidA) is an FAD-containing monooxygenase that catalyzes the hydroxylation of ornithine in the biosynthesis of ...

科研

生物工程

Detection of Total Reactive Oxygen Species in Adherent Cells by 2',7'-Dichlorodihydrofluorescein Diacetate Staining

Oxidative stress is an important event under both physiological and pathological conditions. In this study, we demonstrate how to quantify oxidative stress by measuring total reactive oxygen species (ROS) using 2&#39;,7&#39;-dichlorodihydrofluorescein diacetate (DCFH-DA) staining in colorectal cancer cell lines as an example. This protocol describes detailed steps including preparation of DCFH-DA solution, incubation of cells with DCFH-DA solution, and measurement of normalized intensity. DCFH-DA staining is a simple and cost-effective way to detect ROS in cells. It can be used to measure ROS generation after chemical treatment or genetic modifications. Therefore, it is useful for determining cellular oxidative stress upon environment stress, providing clues to mechanistic studies.

Here, we present a protocol to detect total cellular reactive oxygen species (ROS) using 2&#39;,7&#39;-dichlorodihydrofluorescein diacetate (DCFH-DA). This method can visualize cellular ROS localization in adherent cells with a fluorescence microscope and quantify ROS intensity with a fluorescence plate reader. This protocol is simple, efficient and cost-effective.

通过2'，7'-二氯二氢氟辛二乙酸染色检测粘附细胞中的总活性氧物种

Oxidative stress is an important event under both physiological and pathological conditions. In this study, we demonstrate how to quantify oxidative ...

生物化学

稳定同位素追踪和LC-MS分析以测量DHODH和SDH活性

Oxygen-Independent Assays to Measure Mitochondrial Function in Mammals

The flow of electrons in the mitochondrial electron transport chain (ETC) supports multifaceted biosynthetic, bioenergetic, and signaling functions in mammalian cells. As oxygen (O2) is the most ubiquitous terminal electron acceptor for the mammalian ETC, the O2 consumption rate is frequently used as a proxy for mitochondrial function. However, emerging research demonstrates that this parameter is not always indicative of mitochondrial function, as fumarate can be employed as an alternative electron acceptor to sustain mitochondrial functions in hypoxia. This article compiles a series of protocols that allow researchers to measure mitochondrial function independently of the O2 consumption rate. These assays are particularly useful when studying mitochondrial function in hypoxic environments. Specifically, we describe methods to measure mitochondrial ATP production, de novo pyrimidine biosynthesis, NADH oxidation by complex I, and superoxide production. In combination with classical respirometry experiments, these orthogonal and economical assays will provide researchers with a more comprehensive assessment of mitochondrial function in their system of interest.

作者聚焦：测量哺乳动物线粒体功能的氧非依赖型检测  

Here, we present a compilation of assays to directly measure mitochondrial function in mammalian cells independently of their ability to consume molecular oxygen.

测量哺乳动物线粒体功能的氧非依赖性测定

The flow of electrons in the mitochondrial electron transport chain (ETC) supports multifaceted biosynthetic, bioenergetic, and signaling functions in ...

Mitochondrial Functional Assessment in Brush Lizard Liver Mitochondria

Assessment of Mitochondrial Oxygen Consumption Using a Plate Reader-based Fluorescent Assay

Mitochondria serve many important functions, including cellular respiration, ATP production, controlling apoptosis, and acting as a central hub of metabolic pathways. Therefore, experimentally assessing mitochondrial functionality can provide insight into variations among different populations or disease states. Additionally, it is valuable to assess whether isolated mitochondria are healthy enough to proceed with experiments. One characteristic often used to compare mitochondrial function in different samples is the rate of oxygen consumption. Oxygen consumption and subsequent calculation of the respiratory control ratio in either intact cells or mitochondria isolated from tissue can serve all three purposes. Using mitochondria isolated from the livers of brush lizards in conjunction with a phosphorescent probe that is sensitive to the fluctuations in oxygen concentration of a solution, we measured oxygen consumption using a fluorescent plate reader. This method is not only quick and efficient but also can be conducted with a small amount of mitochondria and without the need for specialized equipment. The step-by-step protocol described here increases the accessibility of mitochondrial functional assessment to researchers.

Assessment of oxygen consumption provides integral information about mitochondrial function. Using a phosphorescent probe with a fluorescent plate reader, accurate and reproducible data can be obtained easily without specialized equipment. This assay enables any lab to measure the oxygen consumption of isolated mitochondria and calculate respiratory control ratios.

Mitochondria serve many important functions, including cellular respiration, ATP production, controlling apoptosis, and acting as a central hub of ...

<meta charset="utf8"></head><body>改变线粒体功能以用于治疗和生物杀虫剂应用

Unveiling Xenobiotic Transport and Effects in Isolated Mitochondria: Insights from Respirometric and Enzymatic Assays

Mitochondria are often referred to as the cell's powerhouse due to their crucial role in energy production through the electron transport chain (ETC). However, their significance extends far beyond energy production. Dysregulation of mitochondrial bioenergetics can trigger intracellular cascades that impact health and cellular function. Given their physiological importance, there is growing interest in exploring the pharmacological potential of mitochondrial function for developing new therapies and bioproducts. Modulating mitochondrial bioenergetics could provide innovative approaches to address challenges such as neurodegenerative disorders, metabolic diseases, and cancer, as well as to develop bioproducts for controlling pests and vector-borne diseases.
This work presents a method for assessing the effects of xenobiotics on the electron transport chain (ETC) using various substrates, including glutamate and NADH for complex I, succinate for complex II, and cytochrome c (both oxidized and reduced) for complexes III and IV. This methodology allows the activation or inhibition of electron transport through mitochondrial complexes to be evaluated using a respirometer and spectrophotometer. The mitochondria can be sourced from isolated mitochondria, fragmented cells, or homogenized tissue from various species.
In the laboratory, mitochondrial function has been analyzed in Aedes aegypti and Wistar rats, but this method is also applicable to other species, such as Rhipicephalus microplus and Rhodnius prolixus. This approach provides the basis for theorizing about the existence of uncoupler proteins and species-specific oxidizable substrate preferences influenced by their unique energetic demands. Recent findings offer valuable insights into innovative bioinsecticide design strategies that target mitochondrial function, holding significant potential for effectively controlling vector-borne diseases and pest infestations.

<meta charset="utf8"></head><body>揭示离体线粒体中的异种生物运输和影响:来自呼吸测定和酶测定的见解

This study introduces a methodology for studying xenobiotic effects on mitochondrial function via respirometric and enzymatic assays. It examines electron transport chain modulation in various species, offering insights for targeted biopesticide development against insect vector-borne diseases.

揭示离体线粒体中的异种生物运输和影响:来自呼吸测定和酶测定的见解

Mitochondria are often referred to as the cell's powerhouse due to their crucial role in energy production through the electron transport chain (ETC). ...

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 ...

含黄素线粒体脱氢酶同时测定超氧化物/过氧化氢和 NADH 生产

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监视依赖黄素单加氧酶和氧化还原半反应，用停流分光光度法

通过2'，7'-二氯二氢氟辛二乙酸染色检测粘附细胞中的总活性氧物种

测量哺乳动物线粒体功能的氧非依赖性测定

测量哺乳动物线粒体功能的氧非依赖性测定

测量哺乳动物线粒体功能的氧非依赖性测定

Assessment of Mitochondrial Oxygen Consumption Using a Plate Reader-based Fluorescent Assay

揭示离体线粒体中的异种生物运输和影响:来自呼吸测定和酶测定的见解

揭示离体线粒体中的异种生物运输和影响:来自呼吸测定和酶测定的见解

揭示离体线粒体中的异种生物运输和影响:来自呼吸测定和酶测定的见解

小鼠离体心肌线粒体通透性转换孔开放中的多参数测量。