这项研究的经费来自国家科学和工程研究理事会 (NSERC、发现补助金) 和大学的赠款给 NP。LHB 希望感谢加拿大卫生研究所 (卫生研究院)、新不伦瑞克省创新基金会 (NBIF) 和蒙古顿大学提供的资助。东隧的工作由加拿大阿尔茨海默病学会、加拿大脑部、 NSERC 、加拿癌基金会、新不伦瑞克省创新基金会、新不伦瑞克省健康研究基金会和大学提供支持。

1. 试剂制备
<ol><li>为组织的解剖和通透准备以下解决方案。<ol>
<li>准备保存解决方案: 2.77 mM CaK2EGTA, 7.23 毫米 k2EGTA, 5.77 毫米 Na2ATP, 6.56 毫米氯化镁2, 20 毫米牛磺酸, 15 毫米 Na2磷酸肌酸, 20 毫米咪唑, 0.5 毫米 dithiothreitol 和50毫米 k MES, pH 7.1 (可存储在-20 摄氏度)。</li>
		<li>准备皂苷溶液: 5 毫克的皂苷在1毫升的保存溶液 (每日准备新鲜)。</li>
	</ol>
</li>
	<li>为呼吸的测量准备以下解答。<ol>
<li>准备呼吸介质: 120 毫米氯化钾, 5 毫米2PO4, 3 毫米 HEPES, 1 毫米 EGTA, 1 毫米氯化镁2, 0.2% BSA (w/v), pH 7.2。</li>
		<li>准备基质, uncoupler, 抑制剂。将 h2o 中的所有基底溶解, 并溶解 uncoupler FCCP 以及乙醇绝对 (在 H2O 中稀释的丙二酸除外) 中的抑制剂。中和大部分基底和丙二酸 (见材料表)。该协议中所给出的所有浓度都是2毫升腔内的最终浓度。</li>
	</ol>
</li>
</ol>2. 高分辨率呼吸的制备和标定。
<ol><li>用70% 乙醇和三倍的蒸馏水清洗呼吸的腔室、瓶盖和塞子三次。</li>
	<li>在零氧浓度下校准: 吸管2.3 毫升的呼吸介质, 并加入10毫克硫酸钠钠。虽然最大体积的房间是2毫升, 增加2.3 毫升, 以确保没有气泡停留在塞子时关闭室。用塞子关闭腔室, 测量氧气浓度为10-20 分钟. 在零氧浓度下校准电极, 产生耗氧率。</li>
	<li>用馏分水冲洗房间, 在绝对乙醇中孵化10分钟, 然后用蒸馏水冲洗。</li>
	<li>在空气饱和度校准: 吸管2.3 毫升的呼吸介质在房间里, 插入塞子, 吸入多余的介质在塞子顶部, 然后举起塞子与间隔。过量的呼吸介质在腔室中 pipetted (总体积为2毫升), 以避免在关闭腔室后出现气泡。</li>
	<li>监测氧气消耗量为45分钟到一个小时, 直到氧气浓度 (蓝色踪影) 是稳定的大约250毫微米在24°c。</li>
</ol>3. 蝇的解剖和组织的通透
<ol><li>确保所有步骤都在冰上执行。</li>
	<li>麻醉六只雄性苍蝇的同一株, 同一年龄, 并提出在相同的饮食在冰上, 以便于解剖苍蝇 (野生型 w1118, 15 天, 喂食玉米粉培养基)。</li>
	<li>使用手术刀和细尖钳, 解剖苍蝇 (除去头部和腹部), 只保留包含飞行肌肉的胸部。这一步可以用肉眼来完成。用细尖钳处理产生的胸部。</li>
	<li>转移三解剖胸部在25毫米培养皿中含有2毫升的冰冷保存溶液使用镊子。</li>
	<li>用细尖钳, 机械 permeabilize 胸部通过插入钳位到胸部, 并反复撕裂组织, 以获得一个松散连接的网络。</li>
	<li>在24井板中, 用12.5 µL 皂苷溶液和1毫升保存液填充两个井, 以获得最终浓度为62.5 µg/毫升的皂苷。用1毫升的呼吸介质填充两个相邻的井。</li>
	<li>用细尖钳, 将三透胸部转移到稀释的皂苷溶液中, 在冰上的轨道振动筛上进行轻度搅拌, 20 分钟。</li>
	<li>后孵化后, 将纤维转移到相邻的井中, 用温和的搅拌, 在冰上填充呼吸介质, 5 分钟冲洗掉皂苷。</li>
</ol>4. 干重的测定
<ol><li>干燥透胸部在吸收表面和翻转3-4 次使用细尖钳, 以确保所有的水分被删除。</li>
	<li>把三胸部在天平上。注意得到的重量, 因为它将用于正常化线粒体氧消耗率。</li>
	<li>立即在冰上的一滴呼吸介质中转移组织。</li>
</ol>5. 耗氧率测定
<ol><li>打开呼吸的腔室 (取下塞子), 并将10毫米的丙酮酸盐和2毫米的果酸加入每个腔室。</li>
	<li>将透组织直接添加到充满呼吸介质的腔室中。</li>
	<li>用垫片替换塞子。</li>
	<li>用60毫升注射器, 直接从氧气罐中收集氧气, 通过每个腔室的塞子毛细管注入2-5 毫升氧气, 确保通过组织的氧气扩散不会限制氧气消耗。</li>
	<li>将哈密尔顿注射器插入每个塞子的毛细管中, 以确保胸部留在室内。</li>
	<li>当氧气浓度约为 400 nM 时, 完全关闭腔室, 在腔内达到空气饱和度 (约160% 空气饱和度) 以上的氧气水平。</li>
	<li>停止并重新启动搅拌器检查房间内的气泡。</li>
	<li>输入以前称重的组织质量, 通过组织质量 (mg) 使呼吸速率正常化, 显示质量特定的耗氧量 (pmol O2消耗的-1. mg-1 )。</li>
	<li>一旦氧气消耗量稳定 (红色踪影), 增加5毫米 ADP 与哈密尔顿注射器。</li>
	<li>每次注射后, 用蒸馏水、70% 乙醇和蒸馏水冲洗注射器。</li>
	<li>一旦氧气消耗再次稳定, 注射5µL 4 毫米的细胞色素 c。</li>
	<li>进行以下基质的顺序注射, 以评估 ETS 不同步骤的线粒体耗氧量:<ol>
<li>添加5µL 2 米脯氨酸。</li>
		<li>添加10µL 1 米琥珀酸。</li>
		<li>添加30µL 1 米 glycerol-3-phosphate。</li>
	</ol>
</li>
	<li>以滴定方式注入 uncoupler FCCP, 步骤为 0.5-1 µM (2 µL 1 毫米溶液), 直到达到最佳浓度,即浓度, 以达到最高的氧气消耗量可能没有抑制 (警告: 参见表材料的)。</li>
	<li>按顺序注入以下抑制剂, 以完全抑制 ETS 中的电子通量, 以测量残余氧消耗 (火箭), 即非呼吸道反应, 如氧化酶反应等:<ol>
<li>添加1µL 1 mM 鱼藤酮 (注意: 请参阅材料表)。</li>
		<li>添加5µL 2 米丙二酸 (小心: 请参阅材料表)。</li>
		<li>添加1µL 5 毫米 antimycin a (注意: 请参阅材料表)。</li>
	</ol>
</li>
	<li>将0.2 毫米抗坏血酸和0.5 毫米 TMPD 依次添加到腔室中, 从抗坏血酸开始测量复合 iv 的耗氧量。</li>
	<li>添加20毫米叠氮化钠以抑制复杂 IV (注意: 请参阅材料表)。</li>
	<li>信号稳定后, 用间隔打开腔室, 通过注入2毫升纯氧, 并在浓度约为 250-300 nmol 的情况下关闭腔室, 重新氧腔. mL-1。</li>
	<li>测量信号10-15 分钟, 并计算化学背景,即,由于 TMPD 氧化的氧消耗量。</li>
</ol>6. 清洁呼吸
<ol><li>测量后, 用蒸馏水冲洗腔室一次, 在70% 乙醇中冲洗三次腔。</li>
	<li>在绝对乙醇中孵育10分钟, 使抑制剂失效。</li>
	<li>用蒸馏水冲洗三次, 用70% 乙醇填满腔室, 并将塞子和瓶盖更换, 直到下一次使用。</li>
</ol>

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作者没有什么可透露的。

本文介绍了果蝇线粒体耗氧量测量前的样品制备方法。这种方法是为了克服与使用线粒体隔离有关的协议的不同问题, 特别是在持续时间和需要的个人数量方面。与线粒体隔离通常需要大量的组织从几个个体获得, 这个实验是对透肌纤维从胸部的几个果蝇。在本协议中, 只有三个人需要显示最佳结果。因此, 在不同的时间点测量线粒体耗氧量是可能的, 因为同步一小群具有相同年龄和相同性别的苍蝇是可行的。
首先, 在相同的条件下使用相同年龄、性别、应变和凸起的果蝇, 在结果中获得低变异性是很重要的 (图 6), 因为呼吸可以随着年龄、性别、基因型和果蝇的饮食而改变。执行此协议的最关键步骤是胸部的通透过程。这是非常重要的, 以避免组织的退化, 并确保胸部是很好的透有最大的耗氧率, 这也是非常重要的。为了确保通透已正确执行, 并且在通透后保持线粒体的结构和功能完整性, OXPHOS 耦合率 (ci-OXPHOS/ci 泄漏,图 3) 以及对细胞色素 c (图 4) 的响应被用作质量控制。在快速工作和确保通透执行良好之间需要考虑权衡。在操纵解剖胸部的时候, 总是在冰上工作也是至关重要的。为了获得最可重现的结果 (图 6), 通透是必需的, 并且练习通透的技术有助于确保重复相同的动作。另一个关键步骤是测定样品的干重。事实上, 在胸部孵化后, 它们是非常敏感、脆弱和容易退化的。因此, 类似于通透, 快速进行测定干重是很重要的。另一方面, 重要的是确定的重量与准确性, 因为结果是规范化的组织质量。使用的胸部数量也可以优化。该协议可以适应使用一个单一的胸部每室35, 但一定程度的分辨率可能会失去在测量氧气消耗, 就像在测定干重。如果必要的话, 胸部的数量可以增加, 但重要的是要注意到, 样品中的线粒体越多, 氧气消耗和分庭的 reoxygenated 就越高。这种复氧可以通过打开腔室和注入氧气来完成, 但它会增加在腔内插入气泡的可能性。因此, 如果可能的话, 尽量避免不得不 reoxygenate 的房间 (除非在复杂 IV 最大呼吸能力必须确定)。在进行实验之前, 对基质浓度的优化也是必要的, 以达到最高的线粒体氧消耗, 而不观察由于过量浓度的抑制效应。呼吸的分庭也必须彻底清洗, 以避免潜在的污染物。重要的是开始清洗与馏分水, 然后70% 乙醇, 以摆脱潜在的生物污染物。在此之后, 建议在绝对乙醇中孵化10分钟, 以避免疏水性抑制剂的污染。最后, 用馏分水冲洗房间, 用70% 乙醇填充, 以避免潜在的污染, 直到下次使用。
该协议可以很容易地修改, 以研究其他基质或不同抑制剂的效果。例如, 一个有趣的蛋白质研究是线粒体丙酮酸载体 (MPC)。这种蛋白的作用是把丙酮酸在细胞质中产生的线粒体基质。最近, 有人建议在病理条件下, 如2型糖尿病38期间, 对新陈代谢的调节发挥重要作用。为了研究蛋白质 MPC 对线粒体代谢的影响, 只有用丙酮酸盐和苹果酸同时启动注射, 才能有效地进行。因此, 丙酮酸的作用是独立的研究与苹果酸的作用, 因为后者被注射只支持 tricarboxylic 酸性循环, 并确保中间体没有耗尽。还可以使用 MPC39抑制剂来评估这种转运器的限制, 而不是丙酮酸的氧化。然后, 可以观察到呼吸的首选燃料源的潜在变化40。如果它与研究无关, 也可以省略一些基板来简化协议。例如, 为了研究复杂 I 的功能障碍, 没有必要使用本协议中提出的所有基板。
此协议提供了一些从体内条件中转移的限制。当呼吸介质高于空气饱和度时, 就会记录线粒体耗氧量的测量结果, 因为注入纯氧可以防止组织内的氧扩散限制在30内。此外, 基质的浓度被注入过量, 这并不反映体内条件。同样重要的是注意到果蝇的年龄、性别、应变和饮食会影响结果。因此, 在解释结果时将考虑这些参数。然而这种方法比使用线粒体隔离测量线粒体氧消耗的方法更接近生理条件, 因为与其他细胞成分的相互作用和线粒体的结构完整性和形态学被保存为31。重要的是要注意的是, 当额外的参数, 如 ROS 生产或线粒体效率 (P/O 比) 必须测量27时, 线粒体隔离更有利。
该方法可用于实现各种目标, 从对线粒体功能障碍的认识和代谢性疾病的潜在机制, 到检测不同化合物对病理性线粒体机能的影响。条件。此外, 它可以用来观察几种环境应力对线粒体代谢的影响, 如饮食或温度的变化。例如, 有可能研究生物活性合成分子对线粒体功能的有效性, 以及对线粒体机能障碍的治疗。该方法也是研究不同时间点线粒体功能的好工具, 对研究衰老过程的基本机制具有重要意义。使用透胸部对线粒体代谢的评估, 将有助于更好地了解线粒体代谢的作用, 当细胞接触到挑战性的条件。

在一个世纪的 1中, 果蝇, 即, 作为基因研究的模型有机体已经被使用了。这一有机体的研究不仅导致了关于性关联遗传的重要基础知识2, 变异率3, 神经系统的发展和细胞命运确定4, 但最近也出现了作为一个研究阿尔茨海默氏症和帕金森症的5,6等多种疾病固有机制的宝贵工具。此外, 研究衰老过程是一种流行的模型, 因为它们可以在很短的时间内大量的增加, 而且寿命短。它们还拥有与人体器官相似的结构, 如心脏、oenocytes (肝细胞样)、脂肪体 (类似肝脏和白色脂肪组织的功能)、胰岛素产生细胞 (相当于β-胰细胞), 以及血淋巴输送代谢产物 (类似脊椎动物的血液)7。此外, 中间代谢的中心途径 (包括胰岛素/胰岛素样生长因子样信号通路和目标的雷帕霉素-TOR 通路) 也高度保守7。基于这些原因, 果蝇最近被利用来描述控制新陈代谢的基本机制, 特别是在人类代谢疾病 (如糖尿病) 的病理条件下, 如8。新陈代谢的一个主要组成部分是线粒体, 它集成了多种途径, 并通过氧化磷酸化过程 (OXPHOS) 执行生命中最重要的生物功能之一 ATP 生产。考虑到它们在机体新陈代谢中的核心作用, 在许多疾病如帕金森氏症的9和阿尔茨海默病的10以及肌萎缩侧索硬化症中, 线粒体功能障碍的介入并不奇怪。11,12. 它们也是老龄化进程的基本决定因素。事实上, 它们是细胞中活性氧种类 (ROS) 的主要生产者, 通过氧化损伤11, 它们可能对高浓度的细胞有害。衰老也与受损或突变的线粒体 DNA 的积累有关13、mitophagy 功能障碍14、15以及线粒体合成16的损害。线粒体也是细胞稳态的关键决定因素, 因为它们可以利用不同的基底来根据营养素17、18的丰度或稀缺性来调整几个细胞功能。
事实上, 饮食中的不同营养素 (碳水化合物、脂质和蛋白质) 在细胞中被消化、吸收和转运。然后在细胞质中转换它们, 导出的基板被传送到线粒体矩阵中, 它们产生减少当量, 如 NADH 和 FADH219。这些还原当量然后由电子运输系统 (ETS) 的不同的酶配合物氧化。这些复合物嵌入线粒体内膜, 如复合 I 和复杂 ii。此外, 其他酶配合物, 如线粒体 glycerol-3-phosphate 脱氢酶和脯氨酸脱氢酶代表电子进入 ETS20,21的备选路线。这些 "替代" 配合物在昆虫中尤为重要, 根据物种的不同, 它们可以积极参与增加呼吸作用20,22,23,21。电子从这些 ETS 哺养的系统被转移到泛醌和随后到复杂 III, 然后到复杂 IV, 直到最后的接受人, 分子氧气。这种电子转移产生一个质子动力的力量横跨内线粒体膜驱动磷酸化的 ADP 到 ATP 在复杂 V (图 1)。考虑到线粒体在细胞稳态中的中心作用, 使用相关的模型D.腹肌研究线粒体代谢是描述各种病理生理学基础机制的有力工具。条件或在细胞和环境压力下。然而令人惊讶的是, 只有少数的研究实际测量了果蝇的线粒体呼吸24,25,26。事实上, 旨在评估线粒体氧消耗的实验需要分离线粒体。虽然对不同线粒体功能的测量 (如 ROS 生产或 P/O 比作为线粒体效率标记的指标有好处27,28), 这些隔离通常需要相当大数量来自多个个人的组织24,29。对大量组织和个人的这一要求是一个重要的限制因素, 特别是考虑到所有的人应该是相同的年龄, 最好是同一性别的实验, 使呼吸的措施在不同的时间点费力的最好。此外, 线粒体隔离可以对调节线粒体代谢的基本机制提供重要的洞察力, 而用于分离线粒体的方法有几个缺点, 例如难以获得可复制的结果。, 破坏线粒体网络, 改变线粒体结构和功能29,30,31。
本研究的目的是提出一个健壮的协议, 以测量在果蝇的线粒体耗氧量只使用少量的组织从很少的人。该协议包括测量线粒体耗氧量就地使用透肌纤维29从果蝇胸部结合高分辨率 respirometry32,33,34,35. 与传统的线粒体分离方法相比, 该方法具有更多的优越性, 因为与细胞其他成分的相互作用以及线粒体结构和功能在透中的保存更明显。光纤29,31,36, 这使得这种方法在生理上更相关。有了这个协议, 线粒体功能可以准确地评估使用高分辨率 respirometry 在只有三胸部的果蝇, 与基质, 以确定氧消耗量的几个不同的步骤, ETS。因此, 这项议定书可以帮助回答关键问题的基本机制, 控制新陈代谢的环境或病理生理条件下, 利用果蝇模型。
为了测量 ETS 的几个不同步骤的耗氧量, 并评估不同基质对呼吸的贡献, 不同的基质 (图 1)、uncoupler 和抑制剂在通透后使用30 。组织。具体来说, 通过不同的基底进行连续添加, 以刺激电子通过 ETS 的不同配合物进入。uncoupler, 羰基氰化物 4-三氟甲氧基苯腙 (FCCP), 然后添加在最佳浓度, 以测量非耦合呼吸,即, 非磷酸化呼吸刺激到最大的氧气消耗。然后, 对复合物 I、II 和 III 的顺序抑制, 以监测非 ETS 氧化反应引起的残余耗氧量。最后, 用 n、n、n、n、-四甲基对苯二胺 (TMPD)、人工电子提供剂和抗坏血酸的注射来评价复合 IV 最大呼吸能力。值得注意的是, 实验是在 24 °C 上进行的, 因为它是产生苍蝇的温度。

<table><tbody><tr><td>High-resolution respirometer Oxygraph O2K</td><td>Oroboros Instruments, Innsbruck, Austria</td><td>10022-02</td><td>Startup O2K respirometer kit&lt;br/&gt; &amp;nbsp;</td></tr><tr><td>O2K-Titration Set&amp;nbsp;</td><td>Oroboros Instruments, Innsbruck, Austria</td><td>20820-03</td><td>Hamilton syringes with different volumes&lt;br/&gt; &amp;nbsp;</td></tr><tr><td>Datlab software</td><td>Oroboros Instruments, Innsbruck, Austria</td><td>20700</td><td>Software for data acquisition and analysis&lt;br/&gt; &amp;nbsp;</td></tr><tr><td>Fine-tipped antimagnetic forceps</td><td>VWR</td><td>82027-400</td><td>&lt;br/&gt; &amp;nbsp;</td></tr><tr><td>Secura225D-1S-DQE</td><td>Sartorius AG, Goettingen, Germany</td><td></td><td>Semi-micro balance (distributed by several companies)&amp;nbsp;&lt;br/&gt; &amp;nbsp;</td></tr><tr><td>&lt;em&gt;Drosophila melanogaster&lt;/em&gt; wild-type w&lt;sup&gt;1118&lt;/sup&gt;</td><td>Bloomington Drosophila stock Center, IN, USA</td><td></td><td>&lt;br/&gt; Storage Condition: 24 &amp;deg;C</td></tr><tr><td>Ethylene glycol-bis(2-aminoethylether)-&lt;em&gt;N&lt;/em&gt;,&lt;em&gt;N&lt;/em&gt;,&lt;em&gt;N&lt;/em&gt;&amp;prime;,&lt;em&gt;N&lt;/em&gt;&amp;prime;-tetraacetic acid</td><td>Sigma-Aldrich</td><td>E4378</td><td>EGTA&lt;br/&gt; Storage Condition: RT</td></tr><tr><td>KOH</td><td>Sigma-Aldrich</td><td>P1767</td><td>CAUTION: corrosive to metals, acute toxicity, skin corrosion, serious eye damage, acute aquatic toxicity.&lt;br/&gt; Storage Condition: RT</td></tr><tr><td>CaCO&lt;sub&gt;3&lt;/sub&gt;</td><td>Sigma-Aldrich</td><td>C4830</td><td>&lt;br/&gt; Storage Condition: RT</td></tr><tr><td>Na&lt;sub&gt;2&lt;/sub&gt;ATP</td><td>Sigma-Aldrich</td><td>A2383</td><td>&lt;br/&gt; Storage Condition: -20 &amp;deg;C</td></tr><tr><td>MgCl&lt;sub&gt;2&lt;/sub&gt;.6H&lt;sub&gt;2&lt;/sub&gt;O</td><td>Sigma-Aldrich</td><td>M9272</td><td>&lt;br/&gt; Storage Condition: RT</td></tr><tr><td>Taurine</td><td>Sigma-Aldrich</td><td>T0625</td><td>&lt;br/&gt; Storage Condition: RT</td></tr><tr><td>Na&lt;sub&gt;2&lt;/sub&gt;Phosphocreatine</td><td>Sigma-Aldrich</td><td>P7936</td><td>&lt;br/&gt; Storage Condition: -20 &amp;deg;C</td></tr><tr><td>Imidazole</td><td>Sigma-Aldrich</td><td>I5513</td><td>&lt;br/&gt; Storage Condition: RT</td></tr><tr><td>Dithiothreitol</td><td>Sigma-Aldrich</td><td>D0632</td><td>&lt;br/&gt; Storage Condition: 2-8 &amp;deg;C</td></tr><tr><td>MES hydrate</td><td>Sigma-Aldrich</td><td>M8250</td><td>&lt;br/&gt; Storage Condition: RT</td></tr><tr><td>Saponin from quillaja bark</td><td>Sigma-Aldrich</td><td>S7900</td><td>Saponin&lt;br/&gt; Storage Condition: RT&lt;br/&gt; Solution Preparation: 5 mg in 1 mL of preservation solution. Prepare fresh daily.</td></tr><tr><td>KCl</td><td>Sigma-Aldrich</td><td>P9541</td><td>&lt;br/&gt; Storage Condition: RT</td></tr><tr><td>KH&lt;sub&gt;2&lt;/sub&gt;PO&lt;sub&gt;4&lt;/sub&gt;</td><td>Sigma-Aldrich</td><td>P9791</td><td>&lt;br/&gt; Storage Condition: RT</td></tr><tr><td>HEPES</td><td>Sigma-Aldrich</td><td>H3375</td><td>&lt;br/&gt; Storage Condition: RT</td></tr><tr><td>BSA</td><td>Sigma-Aldrich</td><td>05470</td><td>&lt;br/&gt; Storage Condition: 2-8 &amp;deg;C</td></tr><tr><td>Na&lt;sub&gt;2&lt;/sub&gt;S&lt;sub&gt;2&lt;/sub&gt;O&lt;sub&gt;4&lt;/sub&gt;</td><td>Sigma-Aldrich</td><td>157953</td><td>Sodium dithionite. CAUTION: self-heating substances and mixtures, acute toxicity, acute aquatic toxi chronic aquatic toxicity.&lt;br/&gt; Storage Condition: RT</td></tr><tr><td>Sodium pyruvate</td><td>Sigma-Aldrich</td><td>P2256</td><td>Pyruvate&lt;br/&gt; Storage Condition: 2-8 &amp;deg;C&lt;br/&gt; Solution Preparation: In MilliQ water. Prepare fresh daily.</td></tr><tr><td>L-(-)-Malic acid</td><td>Sigma-Aldrich</td><td>M1000</td><td>Malate&lt;br/&gt; Storage Condition: RT&lt;br/&gt; Solution Preparation: In MilliQ water. Neutralize with KOH and store at -20 &amp;deg;C.</td></tr><tr><td>Adenosine 5&#039;-diphosphate monopotassium salt hydrate</td><td>Sigma-Aldrich</td><td>A5285</td><td>ADP&lt;br/&gt; Storage Condition: -20 &amp;deg;C&lt;br/&gt; Solution Preparation: In MilliQ water. Neutralize with KOH and store at -80 &amp;deg;C.</td></tr><tr><td>Cytochrome c from equine heart</td><td>Sigma-Aldrich</td><td>C7752</td><td>Cytochrome c&lt;br/&gt; Storage Condition: -20 &amp;deg;C&lt;br/&gt; Solution Preparation: In MilliQ water. Store at -20 &amp;deg;C.</td></tr><tr><td>L-Proline</td><td>Sigma-Aldrich</td><td>P0380</td><td>Proline&lt;br/&gt; Storage Condition: RT&lt;br/&gt; Solution Preparation: In MilliQ water. Store at -20 &amp;deg;C.</td></tr><tr><td>Sodium succinate dibasic hexahydrate</td><td>Sigma-Aldrich</td><td>S2378</td><td>Succinate&lt;br/&gt; Storage Condition: RT&lt;br/&gt; Solution Preparation: In MilliQ water. Neutralize with HCl and store at -20 &amp;deg;C.</td></tr><tr><td>sn-Glycerol 3-phosphate bis(cyclohexylammonium) salt</td><td>Sigma-Aldrich</td><td>G7886</td><td>Glycerol-3-phosphate&lt;br/&gt; Storage Condition: -20 &amp;deg;C&lt;br/&gt; Solution Preparation: In MilliQ water. Neutralize with HCl and store at -80 &amp;deg;C.</td></tr><tr><td>Carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone</td><td>Sigma-Aldrich</td><td>C2920</td><td>FCCP. CAUTION: acute toxicity, skin sensitisation, chronic aquatic toxicity.&lt;br/&gt; Storage Condition: RT&lt;br/&gt; Solution Preparation: In absolute ethanol. Store in glass vials at -20 &amp;deg;C.</td></tr><tr><td>Rotenone</td><td>Sigma-Aldrich</td><td>R8875</td><td>CAUTION: acute toxicity, skin irritation, eye irritation, specific target organ toxicity (respir sytem), acute aquatic toxicity, chronic aquatic toxicity.&lt;br/&gt; Solution Preparation: In absolute ethanol. Store in dark vials at -20 &amp;deg;C.</td></tr><tr><td>Malonic acid</td><td>Sigma-Aldrich</td><td>M1296</td><td>Malonate. CAUTION: acute toxicity, serious eye damage.&lt;br/&gt; Storage Condition: RT&lt;br/&gt; Solution Preparation: In MilliQ water. Neutralize with KOH. Prepare fresh daily.</td></tr><tr><td>Antimycin A from &lt;em&gt;Streptomyces sp&lt;/em&gt;.</td><td>Sigma-Aldrich</td><td>A8674</td><td>Antimycin A. CAUTION: acute toxicity, acute aquatic toxicity, chronic aquatic toxicity.&lt;br/&gt; Storage Condition: -20 &amp;deg;C&lt;br/&gt; Solution Preparation: In absolute ethanol. Store at -20 &amp;deg;C.</td></tr><tr><td>&lt;em&gt;N&lt;/em&gt;,&lt;em&gt;N&lt;/em&gt;,&lt;em&gt;N&lt;/em&gt;&amp;prime;,&lt;em&gt;N&lt;/em&gt;&amp;prime;-Tetramethyl-&lt;em&gt;p&lt;/em&gt;-phenylenediamine</td><td>Sigma-Aldrich</td><td>T7394</td><td>TMPD&lt;br/&gt; Storage Condition: RT&lt;br/&gt; Solution Preparation: In MilliQ water. Store in dark vials at&amp;nbsp; -20 &amp;deg;C.</td></tr><tr><td>(+)-Sodium L-ascorbate</td><td>Sigma-Aldrich</td><td>A4034</td><td>Ascorbate&lt;br/&gt; Storage Condition: RT&lt;br/&gt; Solution Preparation: In MilliQ water. Store in dark vials at&amp;nbsp; -20 &amp;deg;C.</td></tr><tr><td>NaN&lt;sub&gt;3&lt;/sub&gt;</td><td>Sigma-Aldrich</td><td>S2002</td><td>Sodium azide. CAUTION: acute toxicity (oral and dermal), specific target organ toxicity (brain), aquatic toxicity, chronic aquatic toxicity.&amp;nbsp;&lt;br/&gt; Solution Preparation: In MilliQ water. Store at -20 &amp;deg;C.</td></tr></tbody></table>

measurement of mitochondrial oxygen consumption in permeabilized fibers of drosophila using minimal amounts of tissue

果蝇是一种新兴的代谢模型, 它是一种新的, 它是一种用于研究新陈代谢的模式. 事实上, 果蝇的结构与人体器官是同源的, 具有高度保守的代谢通路, 而且寿命相对较短, 可以在短时间内研究不同的基本机制。然而, 令人惊讶的是, 细胞代谢必不可少的机制之一, 线粒体呼吸, 尚未深入研究的模式。这很可能是因为果蝇线粒体呼吸的测量通常需要很大数量的个体, 得到的结果并不高重现性。在这里, 一个方法, 允许精确测量线粒体氧消耗量使用最小数量的组织从果蝇。在这种方法中, 胸部被解剖和透的机械与尖锐的钳和化学与皂苷, 允许不同的化合物, 以跨细胞膜和调节线粒体呼吸。在通透之后, 执行一个协议来评估电子运输系统的不同配合物对不同基质的氧化能力, 以及它们对 uncoupler 和几种抑制剂的反应。这种方法与使用线粒体隔离的方法相比有许多优点, 因为线粒体仍与其他细胞成分相互作用, 线粒体形态学得到保护, 因此在生理上有相关性。此外, 样品的制备速度更快, 获得的结果具有很高的重现性。通过将果蝇的优势与线粒体呼吸的评价结合起来研究代谢模型, 可以揭示出重要的新见解, 特别是当苍蝇经历不同的环境或病理生理时。条件。

在图 2中提供了使用上面描述的协议进行线粒体耗氧量的代表性跟踪。与透肌纤维一起注射的丙酮酸盐和果酸被称为 CI 泄漏呼吸,即, 当 ETS 的复杂 i 被刺激的 NADH 通过氧化丙酮酸盐和果酸通过tricarboxylic 酸循环 (CI)。在这种呼吸速率期间, 线粒体氧主要维持以补偿质子泄漏,即, 质子从膜间隙空间到线粒体基质, 当 ATP 合成酶不活跃 (泄漏) 30.当添加 adp 时, ATP 合成酶被激活, 电子从复 I 转移到复 IV, 同时磷酸化 adp 为 ATP (CI-OXPHOS), 导致线粒体氧消耗增加。OXPHOS 耦合比被计算为 ci-OXPHOS/ci 泄漏, 通常作为一个良好的指标线粒体质量和线粒体耦合30。在果蝇中, 此比例至少应超过 6.0 (图 3)。如果它低于这个值, 它可能表明组织准备或线粒体功能障碍的问题。添加细胞色素 c 可以确定外线粒体膜的完整性, 因此被用作制剂的质量控制 (图 4)。事实上, 细胞色素 c 与内线粒体膜松散绑定, 如果外层线粒体膜在通透过程中受损, 则通常会被冲走。因此, 添加外源细胞色素 c 将显著增加氧气消耗, 如果外部线粒体膜受损和内源性细胞色素 c 丢失。在耗氧量中增加少于 10-15% (图 4) 通常说明了外部线粒体膜29的适当完整性。在图 3和4中, 从非适当的通透和处理样本 (对图 3的组织的过度撕裂, 并在图 4的吸收面上较长时间后称重), 获得了绿色痕迹,而红色的痕迹代表了充分透和处理的样本。这些结果突显出适当的通透条件对于可靠地评估线粒体耗氧量是至关重要的。
在实验中使用的下面的基板为泛醌提供电子, 并允许将电子通量增加到 ETS 中。脯氨酸是一种氨基酸, 可作为能源基质, 特别是在昆虫23, 但也在哺乳动物在急性饥饿期间或在病理条件37。在分庭中加入脯氨酸, 可以评估脯氨酸脱氢酶 (ProDH) 对 ETS 电子通量的贡献, 当电子从复杂的 I 和 ProDH 流动, 并参与 OXPHOS 过程 (CI + ProDH OXPHOS)。除琥珀酸外, 复合 II (琥珀酸脱氢酶) 对 ETS 的贡献可以观察 (CI + ProDH + CII-OXPHOS)。注射 glycerol-3-phosphate (G3P), 进一步增加线粒体氧消耗, 因为此基质刺激线粒体 glycerol-3-phosphate 脱氢酶 (G3PDH), 这是甘油穿梭和转移的一部分电子到 ETS (CI+ProDH+CII+G3PDH-OXPHOS)。ProDH 和 G3PDH 都已被证明在几种昆虫中特别活跃, 因此在本协议中与果蝇20,21,22,23,32.
当添加 uncoupler FCCP 时, 将获得非耦合呼吸 (ets 状态),即, 最大耗氧量代表 ETS (CI+ProDH+CII+G3P-ETS) 的最大容量。FCCP 是一种 protonophore, 它将质子从膜间隙空间传输到线粒体基质而不经过复杂的 v。FCCP 必须小心滴定, 因为浓度低于最佳结果的非最大耗氧量和不稳定的呼吸率, 而浓度高于最佳结果抑制线粒体耗氧量 (图 5)。一旦添加了基质和 uncoupler, 就可以依次使用鱼藤酮、丙二酸和 antimycin A 对复合物 I、II 和 III 的抑制作用。用每一个抑制剂, 线粒体氧消耗减少, 直到达到最低的氧气消耗后, 添加 antimycin a。在添加所有抑制剂后达到的速率是剩余耗氧量30。它代表了氧气消耗量由于非线粒体氧化反应例如加氧酶反应和反应种类生产例如, 并且从其他测量的所有率被减去。
TMPD 是一种人工电子运输器, 它提供电子直接到复杂 iv, 绕过所有的配合物上游, 允许测量复杂 IV 最大呼吸能力。然而, 这种基质容易氧化, 所以抗坏血酸必须在 TMPD 之前添加, 以限制但不能完全避免这种氧化。为了纠正 TMPD 的剩余氧化, 将复合 IV 抑制剂叠氮化钠添加到腔室中, 然后将氧气消耗记录10-15 分钟。因此, 这种耗氧量代表了化学背景, 或者说是 TMPD 的氧化, 在计算复杂 IV 最大呼吸能力时应考虑到这一点。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/57376/57376fig1.jpg"> 图 1.线粒体电子输运的示意图, 由电子传输系统和线粒体内膜氧化磷酸化.i: 复杂 i;二: 复杂的 ii;三: 复杂 iii;iv: 复杂 iv;V: ATP 合成酶;乙酰 CoA: 乙酰辅酶 A;Cyt c: 细胞色素 c;e-: 电子;G3P: glycerol-3-phosphate;G3PDH: 线粒体 glycerol-3-phosphate 脱氢酶;H+: 质子;PRODH: 脯氨酸脱氢酶;TCA: tricarboxylic 酸循环。<a href="https://cloudflare.jove.com/files/ftp_upload/57376/57376fig1large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/57376/57376fig2.jpg"> 图 2: 对果蝇透胸部的线粒体氧浓度 (蓝迹) 和消耗量 (红迹) 的代表性痕迹.每一个注射都用一个箭头表示化合物注射 (Pyr: 丙酮酸;adp;Cyt c: 细胞色素 c;Pro: 脯氨酸;Succ: 琥珀酸;G3P: glycerol-3-phosphate;FCCP: 羰基氰化物 4-三氟甲氧基苯腙;腐烂: 鱼藤酮;马洛: 丙二酸;蚂蚁 a: antimycin;TMPD: n, n, n, n-, 四甲基苯二胺;抗坏血酸;SAZ: 叠氮化钠)。当耗氧量 (红迹) 稳定时, 每个线粒体氧耗率均在图上表示。细胞色素 c 效应表示外线粒体膜的完整性 (详见文本)。残余耗氧量代表了细胞中氧化副反应引起的耗氧量, 必须减去所有其他测量的速率。化学背景是指由于 TMPD 的氧化而产生的耗氧量, 必须考虑到复 IV 最大呼吸能力的计算。<a href="https://cloudflare.jove.com/files/ftp_upload/57376/57376fig2large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/57376/57376fig3.jpg"> 图 3: 有代表性的痕迹有效 (红迹, a 室) 和不足 (绿迹, B 室) 对添加 ADP 的反应.红色踪影对应于房间 A, 并且绿色踪影对应于 B 室, 在同样实验期间。从胸部得到充分透的红迹, 而在通透中过度撕裂组织后获得绿迹。当添加 ADP 时, 预期氧气消耗量会增加, 就像红色的痕迹。OXPHOS 耦合比计算为 ci-OXPHOS/ci 泄漏, 并在图中提出。果蝇中小于6.0 的比率表明线粒体的耦合问题, 是样本退化或由绿迹所代表的线粒体功能障碍的特征。Pyr: 丙酮酸;苹果酸。<a href="https://cloudflare.jove.com/files/ftp_upload/57376/57376fig3large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/57376/57376fig4.jpg"> 图 4: 有代表性的痕迹有效 (红迹, 分庭 a) 和不足 (绿迹, B 室) 对添加细胞色素 c 的反应.红色踪影对应于房间 A, 并且绿色踪影对应于 B 室, 在同样实验期间。从胸部得到充分透和处理的红迹, 而在称重前, 胸部在吸水表面经过较长时间的干燥, 获得了绿迹。细胞色素 c 通常不增加线粒体氧消耗, 如在红色的痕迹上看到的, 表明外线粒体膜是完整的。但是, 如果添加外源细胞色素 c 显著增加15% 以上的线粒体耗氧量, 如绿迹所示, 则表明外线粒体膜受损, 因此样品降解, 应丢弃。Pyr: 丙酮酸;玛: 苹果酸;Cyt c: 细胞色素 c.<a href="https://cloudflare.jove.com/files/ftp_upload/57376/57376fig4large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/57376/57376fig5.jpg"> 图 5: FCCP 滴定后线粒体氧浓度 (蓝迹) 和消耗量 (红迹) 的代表性痕迹.必须仔细执行不同浓度的 FCCP 注射, 以确定此 uncoupler 触发的最大耗氧量。浓度不足不允许对最大耗氧量进行评估, 其特征是不稳定的呼吸速率, 而 FCCP 过量抑制线粒体氧消耗。箭指的是不同的 FCCP 注射。<a href="https://cloudflare.jove.com/files/ftp_upload/57376/57376fig5large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 6" class="xfigimg" src="/files/ftp_upload/57376/57376fig6.jpg"> 图 6: 在使用两个不同腔 (红迹、a 室 a; 绿迹、B 室) 的典型实验中, 呼吸的线粒体氧耗率重现性.红色踪影对应于房间 A, 并且绿色踪影对应于 B 室, 在同一实验期间使用果蝇从同样年龄、性、劳损和上升在同样膳食, 并且协议描述。这两种样品均按照《议定书》 (分别为 A 和 B 室的0.53 和0.66 毫克) 所称的干组织质量进行规范化。<a href="https://cloudflare.jove.com/files/ftp_upload/57376/57376fig6large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>

Watch this Scientific Journal Video about Measurement of Mitochondrial Oxygen Consumption in Permeabilized Fibers of Drosophila Using Minimal Amounts of Tissue at JoVE.com

Measurement of Mitochondrial Oxygen Consumption in Permeabilized Fibers of Drosophila Using Minimal Amounts of Tissue

本文介绍了果蝇透胸部高分辨率 respirometry 测量耗氧量的方法。与经典的线粒体分离技术相比, 这种技术需要最少的组织量, 获得的结果与生理相关。

用最小组织量测定果蝇透纤维中线粒体耗氧量

The fruit fly, Drosophila melanogaster, represents an emerging model for the study of metabolism. Indeed, drosophila have structures homologous to human ...

measurement-mitochondrial-oxygen-consumption-permeabilized-fibers

Nanyang Technological University

Research

JoVE Journal

Biochemistry

11.8K 次观看.  Université de Moncton. 本文介绍了果蝇透胸部高分辨率 respirometry 测量耗氧量的方法。与经典的线粒体分离技术相比, 这种技术需要最少的组织量, 获得的结果与生理相关。

视频: Measurement of Mitochondrial Oxygen Consumption in Permeabilized Fibers of Drosophila Using Minimal Amounts of Tissue

Using Isolated Mitochondria from Minimal Quantities of Mouse Skeletal Muscle for High throughput Microplate Respiratory Measurements

Skeletal muscle mitochondria play a specific role in many disease pathologies. As such, the measurement of oxygen consumption as an indicator of mitochondrial function in this tissue has become more prevalent. Although many technologies and assays exist that measure mitochondrial respiratory pathways in a variety of cells, tissue and species, there is currently a void in the literature in regards to the compilation of these assays using isolated mitochondria from mouse skeletal muscle for use in microplate based technologies. Importantly, the use of microplate based respirometric assays is growing among mitochondrial biologists as it allows for high throughput measurements using minimal quantities of isolated mitochondria. Therefore, a collection of microplate based respirometric assays were developed that are able to assess mechanistic changes/adaptations in oxygen consumption in a commonly used animal model. The methods presented herein provide step-by-step instructions to perform these assays with an optimal amount of mitochondrial protein and reagents, and high precision as evidenced by the minimal variance across the dynamic range of each assay.

The methods presented provide step-by-step instructions for the performance of a collection of microplate based respirometric assays using isolated mitochondria from minimal quantities of mouse skeletal muscle. These assays are able to measure mechanistic changes/adaptations in mitochondrial oxygen consumption in a commonly used animal model.

使用分离线粒体从小鼠骨骼肌进行高通量微孔板呼吸测量最小数量

Skeletal muscle mitochondria play a specific role in many disease pathologies. As such, the measurement of oxygen consumption as an indicator of ...

科研

发育生物学

Measuring and Interpreting Oxygen Consumption Rates in Whole Fly Head Segments

Regulated metabolic activity is essential for the normal functioning of living cells. Indeed, altered metabolic activity is causally linked with the progression of cancer, diabetes, neurodegeneration, and aging to name a few. For instance, changes in mitochondrial activity, the cell&#39;s metabolic powerhouse, have been characterized in many such diseases. Generally, the oxygen consumption rates of mitochondria were considered a reliable readout of mitochondrial activity and measurements in some of these studies were based on isolated mitochondria or cells. However, such conditions may not represent the complexity of a whole tissue. Recently, we have developed a novel method that enables the dynamic measurement of oxygen consumption rates from whole isolated fly heads. By utilizing this method, we have recorded lower oxygen consumption rates of the whole head segment in young versus aged flies. Secondly, we have discovered that lysine deacetylase inhibitors rapidly alter the oxygen consumption in the whole head. Our novel technique may therefore aid in uncovering new properties of various drugs, which may impact metabolic rates. Furthermore, our method may give a better understanding of metabolic behavior in an experimental setup that more closely resembles physiological states.

Measuring alterations in metabolic rates is central to understanding the progression of various diseases and aging. Here, we present a novel technique to measure whole head oxygen consumption that more closely resembles the physiological state and may aid in revealing novel drugs that modify mitochondrial activity.

全飞头段耗氧率的测定与解释

Regulated metabolic activity is essential for the normal functioning of living cells. Indeed, altered metabolic activity is causally linked with the ...

生物化学

An Affordable and Efficient "Homemade" Platform for Drosophila Behavioral Studies, and an Accompanying Protocol for Larval Mitochondrial Respirometry

The usefulness of Drosophila as a model organism for the study of human diseases, behaviors and basic biology is unquestionable. Although practical, Drosophila research lacks popularity in developing countries, possibly due to the misinformed idea that establishing a lab and performing relevant experiments with such tiny insects is difficult and requires expensive, specialized apparatuses. Here, we describe how to build an affordable flylab to quantitatively analyze a myriad of behavioral parameters in D. melanogaster, by 3D-printing many of the necessary pieces of equipment. We provide protocols to build in-house vial racks, courtship arenas, apparatuses for locomotor assays, etc., to be used for general fly maintenance and to perform behavioral experiments using adult flies and larvae. We also provide protocols on how to use more sophisticated systems, such as a high resolution oxygraph, to measure mitochondrial oxygen consumption in larval samples, and show its association with behavioral changes in the larvae upon the xenotopic expression of the mitochondrial alternative oxidase (AOX). AOX increases larval activity and mitochondrial leak respiration, and accelerates development at low temperatures, which is consistent with a thermogenic role for the enzyme. We hope these protocols will inspire researchers, especially from developing countries, to use Drosophila to easily combine behavior and mitochondrial metabolism data, which may lead to information on genes and/or environmental conditions that may also regulate human physiology and disease states.

An Affordable and Efficient "Homemade" Platform for Drosophila Behavioral Studies, and an Accompanying Protocol for Larval Mitochondrial Respirometry

We provide protocols for anyone with a "maker culture" mind to start building a flylab for quantitative analysis of a myriad of behavioral parameters in Drosophila melanogaster, by 3D-printing many of the necessary pieces of equipment. We also describe a high resolution respirometry protocol using larvae to combine behavioral and mitochondrial metabolism data.

The usefulness of Drosophila as a model organism for the study of human diseases, behaviors and basic biology is unquestionable. Although practical, ...

行为学

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 ...

分析果蝇的线粒体生理学和生物能量学

Analyzing Mitochondrial Function in a Drosophila melanogaster PINK1B9-Null Mutant Using High-resolution Respirometry

Neurodegenerative diseases, including Parkinson&#39;s Disease (PD), and cellular disturbances such as cancer are some of the disorders that disrupt energy metabolism with impairment of mitochondrial functions. Mitochondria are organelles that control both energy metabolism and cellular processes involved in cell survival and death. For this reason, approaches to evaluate mitochondrial function can offer important insights into cellular conditions in pathological and physiological processes. In this regard, high-resolution respirometry (HRR) protocols allow evaluation of the whole mitochondrial respiratory chain function or the activity of specific mitochondrial complexes. Furthermore, studying mitochondrial physiology and bioenergetics requires genetically and experimentally tractable models such as Drosophila melanogaster.
This model presents several advantages, such as its similarity to human physiology, its rapid life cycle, easy maintenance, cost-effectiveness, high throughput capabilities, and a minimized number of ethical concerns. These attributes collectively establish it as an invaluable tool for dissecting complex cellular processes. The present work explains how to analyze mitochondrial function using the Drosophila melanogaster PINK1B9-null mutant. The pink1 gene is responsible for encoding PTEN-induced putative kinase 1, through a process recognized as mitophagy, which is crucial for the removal of dysfunctional mitochondria from the mitochondrial network. Mutations in this gene have been associated with an autosomal recessive early-onset familial form of PD. This model can be used to study mitochondrial dysfunction involved in the pathophysiology of PD.

作者聚焦：使用黑腹果蝇探索线粒体功能和化学毒性 

Here we present a high-resolution respirometry protocol to analyze bioenergetics in PINK1B9-null mutant fruit flies. The method uses the Substrate-Uncoupler-Inhibitor-Titration (SUIT) protocol.

使用高分辨率呼吸测量法分析 果蝇黑腹 果蝇 PINK1B9-Null 突变体的线粒体功能

Neurodegenerative diseases, including Parkinson&#39;s Disease (PD), and cellular disturbances such as cancer are some of the disorders that disrupt energy ...

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 ...

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

Metabolic Analysis of Drosophila melanogaster Larval and Adult Brains

This protocol describes a method for measuring the metabolism in Drosophila melanogaster larval and adult brains. Quantifying metabolism in whole organs provides a tissue-level understanding of energy utilization that cannot be captured when analyzing primary cells and cell lines. While this analysis is ex vivo, it allows for the measurement from a number of specialized cells working together to perform a function in one tissue and more closely models the in vivo organ. Metabolic reprogramming has been observed in many neurological diseases, including neoplasia, and neurodegenerative diseases. This protocol was developed to assist the D. melanogaster community&#39;s investigation of metabolism in neurological disease models using a commercially available metabolic analyzer. Measuring metabolism of whole brains in the metabolic analyzer is challenging due to the geometry of the brain. This analyzer requires samples to remain at the bottom of a 96-well plate. Cell samples and tissue punches can adhere to the surface of the cell plate or utilize spheroid plates, respectively. However, the spherical, three-dimensional shape of D. melanogaster brains prevents the tissue from adhering to the plate. This protocol requires a specially designed and manufactured micro-tissue restraint that circumvents this problem by preventing any movement of the brain while still allowing metabolic measurements from the analyzer&#39;s two solid-state sensor probes. Oxygen consumption and extracellular acidification rates are reproducible and sensitive to a treatment with metabolic inhibitors. With a minor optimization, this protocol can be adapted for use with any whole tissue and/or model system, provided that the sample size does not exceed the chamber generated by the restraint. While basal metabolic measurements and an analysis after a treatment with mitochondrial inhibitors are described within this protocol, countless experimental conditions, such as energy source preference and rearing environment, could be interrogated.

Metabolic Analysis of Drosophila melanogaster Larval and Adult Brains

We present a protocol for measuring oxygen consumption and extracellular acidification in Drosophila melanogaster larval and adult brains. A metabolic analyzer is utilized with an adapted and optimized protocol. Micro-tissue restraints are a critical component of this protocol and were designed and created specifically for their use in this analysis.

果蝇幼虫和成年脑的代谢分析

This protocol describes a method for measuring the metabolism in Drosophila melanogaster larval and adult brains. Quantifying metabolism in whole organs ...

用最小组织量测定果蝇透纤维中线粒体耗氧量

摘要

探索更多视频

此视频中的章节

使用分离线粒体从小鼠骨骼肌进行高通量微孔板呼吸测量最小数量

全飞头段耗氧率的测定与解释

An Affordable and Efficient "Homemade" Platform for Drosophila Behavioral Studies, and an Accompanying Protocol for Larval Mitochondrial Respirometry

通过高分辨率呼吸测量法评估坐骨神经的线粒体功能

使用高分辨率呼吸测量法分析果蝇黑腹果蝇 PINK1^B9-Null 突变体的线粒体功能

使用高分辨率呼吸测量法分析果蝇黑腹果蝇 PINK1^B9-Null 突变体的线粒体功能

使用高分辨率呼吸测量法分析果蝇黑腹果蝇 PINK1^B9-Null 突变体的线粒体功能

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

小鼠离体心肌线粒体通透性转换孔开放中的多参数测量。

果蝇幼虫和成年脑的代谢分析

用最小组织量测定果蝇透纤维中线粒体耗氧量

摘要

探索更多视频

此视频中的章节

使用分离线粒体从小鼠骨骼肌进行高通量微孔板呼吸测量最小数量

全飞头段耗氧率的测定与解释

An Affordable and Efficient "Homemade" Platform for Drosophila Behavioral Studies, and an Accompanying Protocol for Larval Mitochondrial Respirometry

通过高分辨率呼吸测量法评估坐骨神经的线粒体功能

使用高分辨率呼吸测量法分析 果蝇黑腹 果蝇 PINK1B9-Null 突变体的线粒体功能

使用高分辨率呼吸测量法分析 果蝇黑腹 果蝇 PINK1B9-Null 突变体的线粒体功能

使用高分辨率呼吸测量法分析 果蝇黑腹 果蝇 PINK1B9-Null 突变体的线粒体功能

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

小鼠离体心肌线粒体通透性转换孔开放中的多参数测量。

果蝇幼虫和成年脑的代谢分析

使用高分辨率呼吸测量法分析果蝇黑腹果蝇 PINK1^B9-Null 突变体的线粒体功能

使用高分辨率呼吸测量法分析果蝇黑腹果蝇 PINK1^B9-Null 突变体的线粒体功能

使用高分辨率呼吸测量法分析果蝇黑腹果蝇 PINK1^B9-Null 突变体的线粒体功能