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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T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mitochondria:+isolation,+structure+and+function.">Mitochondria: isolation, structure and function.</a> The Journal of Physiology. 589 (18), 4413-4421 (2011).</li><li>Pichaud, N., Ballard, J. W. O., Tanguay, R. M., Blier, P. U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Thermal+sensitivity+of+mitochondrial+functions+in+permeabilized+muscle+fibers+from+two+populations+of+Drosophila+simulans+with+divergent+mitotypes.">Thermal sensitivity of mitochondrial functions in permeabilized muscle fibers from two populations of Drosophila simulans with divergent mitotypes.</a> American Journal of Physiology - Regulatory, Integrative and Comparative Physiology. 301 (1), R48-R59 (2011).</li><li>Pichaud, N., Ballard, J. W. O., Tanguay, R. M., Blier, P. U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Naturally+occurring+mitochondrial+dna+haplotypes+exhibit+metabolic+differences:+insight+into+functional+properties+of+mitochondria.">Naturally occurring mitochondrial dna haplotypes exhibit metabolic differences: insight into functional properties of mitochondria.</a> Evolution. 66 (10), 3189-3197 (2012).</li><li>Pichaud, N., Messmer, M., Correa, C. C., Ballard, J. W. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Diet+influences+the+intake+target+and+mitochondrial+functions+of+Drosophila+melanogaster+males.">Diet influences the intake target and mitochondrial functions of Drosophila melanogaster males.</a> Mitochondrion. 13 (6), 817-822 (2013).</li><li>Wolff, J. N., Pichaud, N., Camus, M. F., C&#244;t&#233;, G., Blier, P. U., Dowling, D. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evolutionary+implications+of+mitochondrial+genetic+variation:+mitochondrial+genetic+effects+on+OXPHOS+respiration+and+mitochondrial+quantity+change+with+age+and+sex+in+fruit+flies.">Evolutionary implications of mitochondrial genetic variation: mitochondrial genetic effects on OXPHOS respiration and mitochondrial quantity change with age and sex in fruit flies.</a> Journal of Evolutionary Biology. 29 (4), 736-747 (2016).</li><li>Gnaiger, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Capacity+of+oxidative+phosphorylation+in+human+skeletal+muscle.">Capacity of oxidative phosphorylation in human skeletal muscle.</a> The International Journal of Biochemistry &amp; Cell Biology. 41 (10), 1837-1845 (2009).</li><li>Phang, J. M., Donald, S. P., Pandhare, J., Liu, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+metabolism+of+proline,+a+stress+substrate,+modulates+carcinogenic+pathways.">The metabolism of proline, a stress substrate, modulates carcinogenic pathways.</a> Amino Acids. 35 (4), 681-690 (2008).</li><li>Bender, T., Martinou, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+mitochondrial+pyruvate+carrier+in+health+and+disease:+To+carry+or+not+to+carry?.">The mitochondrial pyruvate carrier in health and disease: To carry or not to carry?.</a> Biochimica et Biophysica Acta - Molecular Cell Research. 1863 (10), 2436-2442 (2016).</li><li>Divakaruni, A. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Thiazolidinediones+are+acute,+specific+inhibitors+of+the+mitochondrial+pyruvate+carrier.">Thiazolidinediones are acute, specific inhibitors of the mitochondrial pyruvate carrier.</a> Proceedings of the National Academy of Sciences of the United States of America. 110 (14), 5422-5427 (2013).</li><li>McCommis, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+ancestral+role+for+the+mitochondrial+pyruvate+carrier+in+glucose-stimulated+insulin+secretion.">An ancestral role for the mitochondrial pyruvate carrier in glucose-stimulated insulin secretion.</a> Molecular Metabolism. 5 (8), 602-614 (2016).</li></ol>

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...

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 border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>High-resolution respirometer Oxygraph O2K</td>
			<td>Oroboros Instruments, Innsbruck, Austria</td>
			<td>10022-02</td>
			<td>Startup O2K respirometer kit 
			&#160;</td>
		</tr>
		<tr>
			<td>O2K-Titration Set&#160;</td>
			<td>Oroboros Instruments, Innsbruck, Austria</td>
			<td>20820-03</td>
			<td>Hamilton syringes with different volumes 
			&#160;</td>
		</tr>
		<tr>
			<td>Datlab software</td>
			<td>Oroboros Instruments, Innsbruck, Austria</td>
			<td>20700</td>
			<td>Software for data acquisition and analysis 
			&#160;</td>
		</tr>
		<tr>
			<td>Fine-tipped antimagnetic forceps</td>
			<td>VWR</td>
			<td>82027-400</td>
			<td> 
			&#160;</td>
		</tr>
		<tr>
			<td>Secura225D-1S-DQE</td>
			<td>Sartorius AG, Goettingen, Germany</td>
			<td></td>
			<td>Semi-micro balance (distributed by several companies)&#160; 
			&#160;</td>
		</tr>
		<tr>
			<td>Drosophila melanogaster wild-type w1118</td>
			<td>Bloomington Drosophila stock Center, IN, USA</td>
			<td></td>
			<td> 
			Storage Condition: 24 &#176;C</td>
		</tr>
		<tr>
			<td>Ethylene glycol-bis(2-aminoethylether)-N,N,N&#8242;,N&#8242;-tetraacetic acid</td>
			<td>Sigma-Aldrich</td>
			<td>E4378</td>
			<td>EGTA 
			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. 
			Storage Condition: RT</td>
		</tr>
		<tr>
			<td>CaCO3</td>
			<td>Sigma-Aldrich</td>
			<td>C4830</td>
			<td> 
			Storage Condition: RT</td>
		</tr>
		<tr>
			<td>Na2ATP</td>
			<td>Sigma-Aldrich</td>
			<td>A2383</td>
			<td> 
			Storage Condition: -20 &#176;C</td>
		</tr>
		<tr>
			<td>MgCl2.6H2O</td>
			<td>Sigma-Aldrich</td>
			<td>M9272</td>
			<td> 
			Storage Condition: RT</td>
		</tr>
		<tr>
			<td>Taurine</td>
			<td>Sigma-Aldrich</td>
			<td>T0625</td>
			<td> 
			Storage Condition: RT</td>
		</tr>
		<tr>
			<td>Na2Phosphocreatine</td>
			<td>Sigma-Aldrich</td>
			<td>P7936</td>
			<td> 
			Storage Condition: -20 &#176;C</td>
		</tr>
		<tr>
			<td>Imidazole</td>
			<td>Sigma-Aldrich</td>
			<td>I5513</td>
			<td> 
			Storage Condition: RT</td>
		</tr>
		<tr>
			<td>Dithiothreitol</td>
			<td>Sigma-Aldrich</td>
			<td>D0632</td>
			<td> 
			Storage Condition: 2-8 &#176;C</td>
		</tr>
		<tr>
			<td>MES hydrate</td>
			<td>Sigma-Aldrich</td>
			<td>M8250</td>
			<td> 
			Storage Condition: RT</td>
		</tr>
		<tr>
			<td>Saponin from quillaja bark</td>
			<td>Sigma-Aldrich</td>
			<td>S7900</td>
			<td>Saponin 
			Storage Condition: RT 
			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> 
			Storage Condition: RT</td>
		</tr>
		<tr>
			<td>KH2PO4</td>
			<td>Sigma-Aldrich</td>
			<td>P9791</td>
			<td> 
			Storage Condition: RT</td>
		</tr>
		<tr>
			<td>HEPES</td>
			<td>Sigma-Aldrich</td>
			<td>H3375</td>
			<td> 
			Storage Condition: RT</td>
		</tr>
		<tr>
			<td>BSA</td>
			<td>Sigma-Aldrich</td>
			<td>05470</td>
			<td> 
			Storage Condition: 2-8 &#176;C</td>
		</tr>
		<tr>
			<td>Na2S2O4</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. 
			Storage Condition: RT</td>
		</tr>
		<tr>
			<td>Sodium pyruvate</td>
			<td>Sigma-Aldrich</td>
			<td>P2256</td>
			<td>Pyruvate 
			Storage Condition: 2-8 &#176;C 
			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 
			Storage Condition: RT 
			Solution Preparation: In MilliQ water. Neutralize with KOH and store at -20 &#176;C.</td>
		</tr>
		<tr>
			<td>Adenosine 5&#39;-diphosphate monopotassium salt hydrate</td>
			<td>Sigma-Aldrich</td>
			<td>A5285</td>
			<td>ADP 
			Storage Condition: -20 &#176;C 
			Solution Preparation: In MilliQ water. Neutralize with KOH and store at -80 &#176;C.</td>
		</tr>
		<tr>
			<td>Cytochrome c from equine heart</td>
			<td>Sigma-Aldrich</td>
			<td>C7752</td>
			<td>Cytochrome c 
			Storage Condition: -20 &#176;C 
			Solution Preparation: In MilliQ water. Store at -20 &#176;C.</td>
		</tr>
		<tr>
			<td>L-Proline</td>
			<td>Sigma-Aldrich</td>
			<td>P0380</td>
			<td>Proline 
			Storage Condition: RT 
			Solution Preparation: In MilliQ water. Store at -20 &#176;C.</td>
		</tr>
		<tr>
			<td>Sodium succinate dibasic hexahydrate</td>
			<td>Sigma-Aldrich</td>
			<td>S2378</td>
			<td>Succinate 
			Storage Condition: RT 
			Solution Preparation: In MilliQ water. Neutralize with HCl and store at -20 &#176;C.</td>
		</tr>
		<tr>
			<td>sn-Glycerol 3-phosphate bis(cyclohexylammonium) salt</td>
			<td>Sigma-Aldrich</td>
			<td>G7886</td>
			<td>Glycerol-3-phosphate 
			Storage Condition: -20 &#176;C 
			Solution Preparation: In MilliQ water. Neutralize with HCl and store at -80 &#176;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. 
			Storage Condition: RT 
			Solution Preparation: In absolute ethanol. Store in glass vials at -20 &#176;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. 
			Solution Preparation: In absolute ethanol. Store in dark vials at -20 &#176;C.</td>
		</tr>
		<tr>
			<td>Malonic acid</td>
			<td>Sigma-Aldrich</td>
			<td>M1296</td>
			<td>Malonate. CAUTION: acute toxicity, serious eye damage. 
			Storage Condition: RT 
			Solution Preparation: In MilliQ water. Neutralize with KOH. Prepare fresh daily.</td>
		</tr>
		<tr>
			<td>Antimycin A from Streptomyces sp.</td>
			<td>Sigma-Aldrich</td>
			<td>A8674</td>
			<td>Antimycin A. CAUTION: acute toxicity, acute aquatic toxicity, chronic aquatic toxicity. 
			Storage Condition: -20 &#176;C 
			Solution Preparation: In absolute ethanol. Store at -20 &#176;C.</td>
		</tr>
		<tr>
			<td>N,N,N&#8242;,N&#8242;-Tetramethyl-p-phenylenediamine</td>
			<td>Sigma-Aldrich</td>
			<td>T7394</td>
			<td>TMPD 
			Storage Condition: RT 
			Solution Preparation: In MilliQ water. Store in dark vials at&#160; -20 &#176;C.</td>
		</tr>
		<tr>
			<td>(+)-Sodium L-ascorbate</td>
			<td>Sigma-Aldrich</td>
			<td>A4034</td>
			<td>Ascorbate 
			Storage Condition: RT 
			Solution Preparation: In MilliQ water. Store in dark vials at&#160; -20 &#176;C.</td>
		</tr>
		<tr>
			<td>NaN3</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.&#160; 
			Solution Preparation: In MilliQ water. Store at -20 &#176;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....

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 ...

The overall goal of this procedure is to evaluate mitochondrial respiration in the common fruit fly, Drosophila Melanogaster, using high resolution respirometry.This is achieved by first dissecting the Drosophila, to collect the thorax.The thoraces containing the flight muscles are then carefully permeabilized mechanically, with sharp forceps, and incubated with saponin, for further permeabilization.The thoraces are then transferred into the chambers of a respirometer, and mitochondrial oxygen consumption is measured with a combination of different substrates, uncouplers and inhibitors.Hi, I am Chlo

measurement-mitochondrial-oxygen-consumption-permeabilized-fibers

Research

JoVE Journal

Biochemistry

11.7K 次观看.  Université de Moncton. The overall goal of this procedure is to evaluate mitochondrial respiration in the common fruit fly, Drosophila Melanogaster, using high resolution respirometry.This is achieved by first dissecting the Drosophila, to collect the thorax.The thoraces containing the flight muscles are then carefully permeabilized mechanically, with sharp forceps, and incubated with saponin, for further permeabilization.The thoraces are then transferred into the chambers of a respirometer, and mitochondrial oxyge...

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

Using Scaffold Liposomes to Reconstitute Lipid-proximal Protein-protein Interactions In Vitro

Studies of integral membrane proteins in vitro are frequently complicated by the presence of a hydrophobic transmembrane domain. Further complicating these studies, reincorporation of detergent-solubilized membrane proteins into liposomes is a stochastic process where protein topology is impossible to enforce. This paper offers an alternative method to these challenging techniques that utilizes a liposome-based scaffold. Protein solubility is enhanced by deletion of the transmembrane domain, and these amino acids are replaced with a tethering moiety, such as a His-tag. This tether interacts with an anchoring group (Ni2+ coordinated by nitrilotriacetic acid (NTA(Ni2+)) for His-tagged proteins), which enforces a uniform protein topology at the surface of the liposome. An example is presented wherein the interaction between Dynamin-related protein 1 (Drp1) with an integral membrane protein, Mitochondrial Fission Factor (Mff), was investigated using this scaffold liposome method. In this work, we have demonstrated the ability of Mff to efficiently recruit soluble Drp1 to the surface of liposomes, which stimulated its GTPase activity. Moreover, Drp1 was able to tubulate the Mff-decorated lipid template in the presence of specific lipids. This example demonstrates the effectiveness of scaffold liposomes using structural and functional assays and highlights the role of Mff in regulating Drp1 activity.

Using Scaffold Liposomes to Reconstitute Lipid-proximal Protein-protein Interactions In Vitro

This paper describes a method for assessing the interactions and assemblies of integral membrane proteins in vitro with various partner factors in a lipid-proximal environment.

使用脚手架脂质体重构脂质近端蛋白质 - 蛋白质相互作用<em&gt;体外</em

Studies of integral membrane proteins in vitro are frequently complicated by the presence of a hydrophobic transmembrane domain. Further complicating ...

科研

生物化学

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

It has been reported that mitochondria can contain up to 12 enzymatic sources of reactive oxygen species (ROS). A majority of these sites include flavin-dependent respiratory complexes and dehydrogenases that produce a mixture of superoxide (O2&#9679;-) and hydrogen peroxide (H2O2). Accurate quantification of the ROS-producing potential of individual sites in isolated mitochondria can be challenging due to the presence of antioxidant defense systems&#160;and side reactions that also form O2&#9679;-/H2O2. Use&#160;of nonspecific inhibitors that can disrupt mitochondrial bioenergetics can also compromise measurements by altering&#160;ROS release from other sites of production. Here, we present an easy method for the simultaneous measurement of H2O2 release and nicotinamide adenine dinucleotide (NADH) production by purified flavin-linked dehydrogenases. For our purposes here, we have used purified pyruvate dehydrogenase complex (PDHC) and &#945;-ketoglutarate dehydrogenase complex (KGDHC) of porcine heart origin as examples. This method allows for an accurate measure of native H2O2 release rates by individual sites of production by eliminating other potential sources of ROS and antioxidant systems. In addition, this method allows for a direct comparison of the relationship between H2O2 release and enzyme activity and the screening of the effectiveness and selectivity of inhibitors for ROS production. Overall, this approach can allow for the in-depth assessment of native rates of ROS release for individual enzymes prior to conducting more sophisticated experiments with isolated mitochondria or permeabilized muscle fiber.

Mitochondria contain several flavin-dependent enzymes that can produce reactive oxygen species (ROS). Monitoring ROS release from individual sites in mitochondria is challenging due to unwanted side reactions. We present an easy, inexpensive method for direct assessment of native rates for ROS release using purified flavoenzymes and microplate fluorometry.

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 ...

Measuring Liver Mitochondrial Oxygen Consumption and Proton Leak Kinetics to Estimate Mitochondrial Respiration in Holstein Dairy Cattle

Oxygen consumption, proton motive force (PMF) and proton leak are measurements of mitochondrial respiration, or how well mitochondria are able to convert NADH and FADH into ATP. Since mitochondria are also the primary site for oxygen use and nutrient oxidation to carbon dioxide and water, how efficiently they use oxygen and produce ATP directly relates to the efficiency of nutrient metabolism, nutrient requirements of the animal, and health of the animal. The purpose of this method is to examine mitochondrial respiration, which can be used to examine the effects of different drugs, diets and environmental effects on mitochondrial metabolism. Results include oxygen consumption measured as proton dependent respiration (State 3) and proton leak dependent respiration (State 4). The ratio of State 3 / State 4 respiration is defined as respiratory control ratio (RCR) and can represent mitochondrial energetic efficiency. Mitochondrial proton leak is a process that allows dissipation of mitochondrial membrane potential (MMP) by uncoupling oxidative phosphorylation from ADP decreasing the efficiency of ATP synthesis. Oxygen and TRMP+ sensitive electrodes with mitochondrial substrates and electron transport chain inhibitors are used to measure State 3 and State 4 respiration, mitochondrial membrane PMF (or the potential to produce ATP) and proton leak. Limitations to this method are that liver tissue must be as fresh as possible and all biopsies and assays must be performed in less than 10 h. This limits the number of samples that can be collected and processed by a single person in a day to approximately 5. However, only 1 g of liver tissue is needed, so in large animals, such as dairy cattle, the amount of sample needed is small relative to liver size and there is little recovery time needed.

Here, we share methods for measuring mitochondrial oxygen consumption, a defining concept of nutritional energetics, and proton leak, the primary cause of inefficiency in mitochondrial generation of ATP. These results can account for 30% of the energy lost in nutrient utilization to help evaluate mitochondrial function.

荷尔斯坦奶牛肝线粒体耗氧量和质子泄漏动力学评价线粒体呼吸

Oxygen consumption, proton motive force (PMF) and proton leak are measurements of mitochondrial respiration, or how well mitochondria are able to convert ...

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 ...

Quantification of Subcellular Glycogen Distribution in Skeletal Muscle Fibers using Transmission Electron Microscopy

With the use of transmission electron microscopy, high-resolution images of fixed samples containing individual muscle fibers can be obtained. This enables quantifications of ultrastructural aspects such as volume fractions, surface area to volume ratios, morphometry, and physical contact sites of different subcellular structures. In the 1970s, a protocol for enhanced staining of glycogen in cells was developed and paved the way for a string of studies on the subcellular localization of glycogen and glycogen particle size using transmission electron microscopy. While most analyses interpret glycogen as if it is homogeneously distributed within the muscle fibers, providing only a single value (e.g., an average concentration), transmission electron microscopy has revealed that glycogen is stored as discrete glycogen particles located in distinct subcellular compartments. Here, the step-by-step protocol from tissue collection to the quantitative determination of the volume fraction and particle diameter of glycogen in the distinct subcellular compartments of individual skeletal muscle fibers is described. Considerations on how to 1) collect and stain tissue specimens, 2) perform image analyses and data handling, 3) evaluate the precision of estimates, 4) discriminate between muscle fiber types, and 5) methodological pitfalls and limitations are included.

A modified post-fixation procedure increases the contrast of glycogen particles in tissue. This paper provides a step-by-step protocol describing how to handle the tissue, conduct the imaging, and use stereological methods to obtain unbiased and quantitative data on fiber type-specific subcellular glycogen distribution in skeletal muscle.

使用透射电子显微镜定量骨骼肌纤维中的亚细胞糖原分布

With the use of transmission electron microscopy, high-resolution images of fixed samples containing individual muscle fibers can be obtained. This ...

mtDNA 合成和分布的高通量定量

High-Throughput Image-Based Quantification of Mitochondrial DNA Synthesis and Distribution

The vast majority of cellular processes require a continuous supply of energy, the most common carrier of which is the ATP molecule. Eukaryotic cells produce most of their ATP in the mitochondria by oxidative phosphorylation. Mitochondria are unique organelles because they have their own genome that is replicated and passed on to the next generation of cells. In contrast to the nuclear genome, there are multiple copies of the mitochondrial genome in the cell. The detailed study of the mechanisms responsible for the replication, repair, and maintenance of the mitochondrial genome is essential for understanding the proper functioning of mitochondria and whole cells under both normal and disease conditions. Here, a method that allows the high-throughput quantification of the synthesis and distribution of mitochondrial DNA (mtDNA) in human cells cultured in vitro is presented. This approach is based on the immunofluorescence detection of actively synthesized DNA molecules labeled by 5-bromo-2'-deoxyuridine (BrdU) incorporation and the concurrent detection of all the mtDNA molecules with anti-DNA antibodies. Additionally, the mitochondria are visualized with specific dyes or antibodies. The culturing of cells in a multi-well format and the utilization of an automated fluorescence microscope make it easier to study the dynamics of mtDNA and the morphology of mitochondria under a variety of experimental conditions in a relatively short time.

作者聚焦：基于图像的高通量线粒体 DNA 合成和分布定量  

A procedure for studying the dynamics of mitochondrial DNA (mtDNA) metabolism in cells using a multi-well plate format and automated immunofluorescence imaging to detect and quantify mtDNA synthesis and distribution is described. This can be further used to investigate the effects of various inhibitors, cellular stresses, and gene silencing on mtDNA metabolism.

基于图像的高通量线粒体DNA合成和分布定量

The vast majority of cellular processes require a continuous supply of energy, the most common carrier of which is the ATP molecule. Eukaryotic cells ...

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 ...