Name: measurement of oxygen consumption rates in intact caenorhabditis elegans
Uploaded: 2019-02-23T15:00:00
Description: Watch this Scientific Journal Video about Measurement of Oxygen Consumption Rates in Intact Caenorhabditis elegans at JoVE.com

作者们要感谢凯文·比特曼博士在实验室建立海马 xfp 方面的指导。国家卫生研究院 gm088213 支持这项工作。

注:图 2提供了完整协议的示意图概述。
1. 线虫种群的生长和同步9,10
<ol><li>将所需遗传背景的 l4 幼虫 (如 n2 [野生类型] 和 sel-12 动物) 转移到线虫生长培养基 (ngm) 板 (菜谱见表 1 ), 新播种的大肠杆菌草坪 (op50)11. 每个应变至少使用两个100毫米或三个60毫米板。在适当的生长...

<ol>
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线粒体呼吸是线粒体功能的一个有见地的指标;因此, 能够测量生物系统中的耗氧率, 无论是在体外还是体内, 都是非常有价值的。呼吸计使用基于斑啉的荧光粉检测氧气水平, 这些荧光粉通过氧或依靠产生与氧气压力成正比的电流的安培氧传感器淬火。克拉克电极属于后一类, 在文献中得到了广泛的应用, 特别是在分析线虫的呼吸时。然而, 由于需要较大的样品尺寸和无法一次评估多个样品, ?...

三磷酸腺苷 (atp) 是细胞能量的主要来源, 是由位于线粒体内膜内的电子传输链 (etc) 中的酶在线粒体中产生的。丙酮酸是用于线粒体 atp 生产的一种关键代谢物, 被导入线粒体基质中, 在那里它被脱羧化, 以产生乙酰辅酶 a (coenzyme)。随后, 乙酰 coa 进入柠檬酸循环, 导致烟酰胺腺嘌呤二核苷酸 (nadh) 的产生, 这是一个关键的电子载体分子。当来自 nadh 的电子通过 etc 传递到氧气时, 质子就会在线粒体膜间空间中积累, 从而导致整个膜产生一个电化学梯度。然后, 这些质子将通过这个电化学梯度从膜间空间流回线粒体基质, 通过 atp 合成酶的质子孔, 推动其旋转和 atp1的合成 (图 1)。
线粒体功能不仅限于能量的产生, 而且对钙的稳态、活性氧的清除和凋亡也至关重要, 关键的是将其功能定位在组织健康中 2.线粒体功能可以使用多种检测方法进行评估, 包括但不限于测量线粒体膜电位、atp 和 ros 水平以及线粒体钙浓度的分析。然而, 这些检测提供了线粒体功能的单一快照, 因此可能无法提供线粒体健康的全面视图。由于 atp 生成过程中的耗氧量依赖于无数的连续反应, 因此它是线粒体功能的一个优越指标。有趣的是, 由于线粒体功能障碍3,4,5, 已经观察到氧气消耗率的变化。
活样品的耗氧率 (ocr) 可以通过可大致分为两类的技术来测量: 安培氧传感器和基于斑岩的荧光粉, 它们可以通过氧6淬火.安培氧传感器已被广泛用于测量 ocr 在培养的细胞, 组织, 和模型系统, 如线虫。然而, 含有呼吸计的基于斑林的荧光粉具有以下优点: (1) 它们允许对两个样品进行三式三份的并排比较, (2) 它们需要较小的样本尺寸 (例如, 每口井20头蠕虫, 而在 2000-500只室)7, 和 (3) 呼吸计可以编程做四个不同的复合注射在整个实验运行的理想时间, 无需手动应用。
在该协议中, 描述了使用基于斑林的氧传感呼吸计测量 ocr 在活的, 完整的线虫所涉及的步骤.虽然有一个书面协议的使用大格式, 高吞吐量呼吸计 8, 该协议已被调整为使用更经济友好, 易于访问, 和较小的规模的仪器。该协议对于评估两个菌株之间的 ocr 差异特别有用, 因为在这两个菌株中不需要高通量筛选, 并且其使用过多。

<table>
	<tbody>
		<tr>
			<td>100 mm, 60 mm Petri dishes</td>
			<td>Kord-Valmark Labware Products</td>
			<td>2900, 2901</td>
			<td></td>
		</tr>
		<tr>
			<td>1.5 mL centrifuge tubes</td>
			<td>Globe Scientific</td>
			<td>6285</td>
			<td></td>
		</tr>
		<tr>
			<td>15 mL conical tubes</td>
			<td>Corning</td>
			<td>430791</td>
			<td></td>
		</tr>
		<tr>
			<td>22 &#215; 22 mm coverslip</td>
			<td>Globe Scientific</td>
			<td>1404-10</td>
			<td></td>
		</tr>
		<tr>
			<td>50 mL conical tubes</td>
			<td>Corning</td>
			<td>430829</td>
			<td></td>
		</tr>
		<tr>
			<td>Agar</td>
			<td>Fisher Scientific</td>
			<td>BP1423-2</td>
			<td></td>
		</tr>
		<tr>
			<td>Bacto peptone</td>
			<td>BD, Bacto</td>
			<td>211677</td>
			<td></td>
		</tr>
		<tr>
			<td>Bacto tryptone</td>
			<td>BD, Bacto</td>
			<td>211705</td>
			<td></td>
		</tr>
		<tr>
			<td>Bacto yeast extract</td>
			<td>BD, Bacto</td>
			<td>212705</td>
			<td></td>
		</tr>
		<tr>
			<td>Bleach</td>
			<td>Generic</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Calcium chloride dihydrate (CaCl2&#183;2H2O)</td>
			<td>Fisher Scientific</td>
			<td>C79-500</td>
			<td></td>
		</tr>
		<tr>
			<td>Carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone (FCCP)</td>
			<td>Abcam</td>
			<td>ab120081</td>
			<td></td>
		</tr>
		<tr>
			<td>Cholesterol</td>
			<td>Fisher Scientific</td>
			<td>C314-500</td>
			<td></td>
		</tr>
		<tr>
			<td>Deionized water (dH2O)</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Dimethyl sulfoxide (DMSO)</td>
			<td>Thomas Scientific</td>
			<td>C987Y85</td>
			<td></td>
		</tr>
		<tr>
			<td>Glass Pasteur pipettes</td>
			<td>Krackeler Scientific</td>
			<td>6-72050-900</td>
			<td></td>
		</tr>
		<tr>
			<td>Magnesium sulfate heptahydrate (MgSO4&#183;7H2O)</td>
			<td>Fisher Scientific</td>
			<td>BP213-1</td>
			<td></td>
		</tr>
		<tr>
			<td>Potassium phosphate dibasic (K2HPO4)</td>
			<td>Fisher Scientific</td>
			<td>BP363-1</td>
			<td></td>
		</tr>
		<tr>
			<td>Potassium phosphate monobasic (KH2PO4)</td>
			<td>Fisher Scientific</td>
			<td>P285-500</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium chloride</td>
			<td>Fisher Scientific</td>
			<td>BP358-10</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium hydroxide (NaOH)</td>
			<td>Fisher Scientific</td>
			<td>BP359-500</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium phosphate dibasic anhydrous (Na2HPO4)</td>
			<td>Fisher Scientific</td>
			<td>BP332-1</td>
			<td></td>
		</tr>
		<tr>
			<td>Seahorse XFp Analyzer</td>
			<td>Agilent</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Seahorse XFp FluxPak</td>
			<td>Agilent</td>
			<td>103022-100</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium Azide</td>
			<td>Sigma-Aldrich</td>
			<td>S2002</td>
			<td></td>
		</tr>
	</tbody>
</table>

measurement of oxygen consumption rates in intact caenorhabditis elegans

最佳线粒体功能对健康的细胞活动至关重要, 特别是在神经系统和肌肉等能量需求较高的细胞中。与此相一致的是, 线粒体功能障碍与无数的神经退行性疾病和一般的衰老有关。线虫是阐明线粒体功能的许多复杂问题的有力模型系统。线粒体呼吸是线粒体功能的强大指标, 最近开发的呼吸计为测量细胞呼吸提供了一个最先进的平台。在这个协议中, 我们提供了一种分析活的、完整的线虫的技术。该协议跨越约7天的时间, 包括 (1) 线虫生长和同步的步骤, (2) 制备注入的化合物和探针的水合作用, (3) 药物装载和弹药筒平衡, (4) 制备蠕虫试验板和分析运行, 以及 (5) 实验后数据分析。

利用本文描述的方法, 测定了野生动物的 ocr 和三种不同的 sel-12突变体菌株。sel-12编码了前青霉素 17的线虫矫形器.人预觉素突变是家族性阿尔茨海默病发展过程中最常见的遗传畸变 18.我们的研究表明, 与野生动物相比,自己 12突变动物的线粒体钙含量升高.由于钙失调会导致自12突变体和野生动...

Watch this Scientific Journal Video about Measurement of Oxygen Consumption Rates in Intact Caenorhabditis elegans at JoVE.com

Measurement of Oxygen Consumption Rates in Intact Caenorhabditis elegans

线虫的耗氧率测定

线粒体呼吸对组织的生存至关重要;因此, 耗氧率是线粒体健康的一个很好的指标。在这个协议中, 我们描述了使用一个商业上可用的呼吸计来测量基本和最大耗氧率的活, 完整, 和自由运动的。

Optimal mitochondrial function is critical for healthy cellular activity, particularly in cells that have high energy demands like those in the nervous ...

线粒体功能对细胞平衡至关重要，氧气消耗率是线粒体健康的决定性指标。该协议提供了分析C.elegans中氧气消耗率的详细说明。该协议利用实时移动C.elegans提供线粒体功能的法律测量。提升隔离线粒体需求，这一步骤可能影响线粒体的完整性。首先，将所需遗传背景的L4幼虫转移到线虫生长介质或NGM板上新鲜播种的大肠杆菌草坪上。每个应变至少使用两个 100 mm 或三个 60 mm 板。在适当的生长温度下孵育蠕虫三到四天，或直到板与大量的鸡蛋和麦格虫集中。每100毫米线虫板用大约6毫升的M9缓冲液将鸡蛋和蠕虫从盘子里洗掉。使用玻璃牧场移液器，将鸡蛋和蠕虫转移到每个菌株的15毫升离心管中。在6，180克下旋转这些管三分钟。离心后，吸出M9缓冲液，只保留动物和蛋丸。在每个管中加入三到四毫升的漂白剂溶液，间歇性涡流6分钟。然后，加入M9缓冲液填充每个管，并在6，180克旋转一分钟。吸上一液，用M9重复洗涤三次。洗涤后，将鸡蛋颗粒移动到含有约9毫升M9缓冲液的新鲜15毫升管中。在 20 摄氏度下加热 16 至 48 小时，使新孵化的蠕虫同步。在将这些管子在 6，180 g 下旋转 1.5 分钟后，将同步的 L-1 动物放在单个 NGM 板上的 OP50 新鲜种子草坪上。将板保持在20摄氏度，直到动物在大约42小时后到达L-4幼虫阶段。此时，使用铂金选择将L4幼虫移至OP50种子NGM板，每毫升FUdR含有0.5毫克，以防止产生后代。通过将 200 微升蒸馏水添加到板上的每一个井中，使传感器墨盒水合一，确保传感器探头被淹没。在室温下隔夜存放板。关闭呼吸计界面内的加热元件。将机器存放在 15 摄氏度的培养箱内可降低机器内部的核心温度，并防止动物在检测过程中过热。经过一夜的孵化，移液器取出并丢弃用于滋润传感器探头的蒸馏水，并在每井中用200微升的校准液代替。将 FCCP 库存溶液稀释至蒸馏水中的 100 微摩尔 FCCP。然后，将稀释溶液的 20 微升添加到传感器墨盒中的喷射端口 A 中。将 22 微升 400 毫摩尔钠 azides 添加到传感器墨盒的喷射端口 B 中。现在，打开呼吸仪。在主屏幕上，选择"开始"，将显示模板页面。在模板页上，选择空白"或以前设计的模板。将显示组页面。在组页上，选择井 A 和 H 作为背景井，根据实验计划将剩余的六口井分配到适当的组中。通过单击右下角的箭头访问协议页，并确保选择平衡、基底和注入一和两个按钮。调整基础OCR的读数数以及最大OCR和非线粒体OCR。选择右下角的箭头。将显示加载传感器墨盒板的提示。按照提醒提示屏幕上的说明，确保传感器墨盒板以正确的方向加载。现在，重新测量仪将平衡墨盒。在细胞板的八个孔和周围储液罐中，每口加入200微升M9缓冲液。从每个菌株中挑选大约 100 个蠕虫到非种子 NGM 板上，并允许休息两到三分钟。然后，用M9缓冲液将铂金采摘的端子弄湿，将20岁同步动物放入B型水井到G型，使背景井 A和H空空。现在，单击呼吸仪软件上的"确定"以弹出包含校准缓冲液的板。从呼吸仪上拆下板，将传感器盒留在仪器内。用含有动物的盘子替换卡钳板。加载板并通过撞击关闭车门，然后让检测运行。检测运行完成后，按照屏幕上的提示删除电池板和传感器盒。将闪存驱动器插入 USB 端口以保存运行数据战格式。拆下传感器墨盒，让动物在井底安顿下来约两分钟。将细胞板放在立体解剖显微镜下，计算每孔的动物数量。打开分析软件，然后打开规范化选项卡，将耗氧率与动物数量标准化。在"修改"选项卡下为各个组应用适当的级别，并导出该文件作为棱镜文件进行进一步分析。前五个测量值的平均值是基础 OCR。添加 FCCP 后五个测量值的平均值为最大 OCR。最后，添加亚兹德钠后最后五次测量的平均数是非线粒体呼吸率。由于钙失调会导致线粒体功能改变，因此对sel-12突变体和野生类动物的OCR进行测量，以检查sel-12突变对线粒体功能和健康的影响。野生类动物的底法OCR率一直低于每分钟5个皮毛，每个蠕虫。虽然所有三个菌株的sel-12突变体显示显著升高的OCR，大约每分钟7个皮科，每个蠕虫。加入FCCP后，野生类型的OCR和sel-12突变体如预期的那样增加。野生类动物显示，每只蠕虫每分钟最大OCR约为7个象形虫。而 sel-12 突变体每分钟有大约 10 个象形虫，每个蠕虫。在此测定中使用活体、喂养良好和异步动物，并避免将鸡蛋或尸体转移到测定井中。此外，仪器的内部温度不应超过 25 摄氏度。漂白剂、FCCP、FudR 和酸钠是有毒的。因此，在准备库存溶液和药物装载时，应注意佩戴防护手套。

Research

JoVE Journal

Biology

Measurement of Oxygen Consumption Rates in Intact Caenorhabditis elegans

Isolation and In vitro Activation of Caenorhabditis elegans Sperm

Isolation and In vitro Activation of Caenorhabditis elegans Sperm

分离和<em在体外激活线虫精子

Males and hermaphrodites are the two naturally found sexual forms in the nematode C. elegans. The amoeboid sperm are produced by both males and ...

科研

生物学

Solid Plate-based Dietary Restriction in Caenorhabditis elegans

Solid Plate-based Dietary Restriction in Caenorhabditis elegans

实心板为基础的饮食限制在线虫</em

Reduction of food intake without malnutrition or starvation is known to increase lifespan and delay the onset of various age-related diseases in a wide ...

Time-lapse Microscopy of Early Embryogenesis in Caenorhabditis elegans

Time-lapse Microscopy of Early Embryogenesis in Caenorhabditis elegans

时间推移显微镜早期胚胎发育线虫

Caenorhabditis elegans has often been used as a model system in studies of early developmental processes.  The transparency of the 
embryos, the genetic ...

Basic Caenorhabditis elegans Methods: Synchronization and Observation

Basic Caenorhabditis elegans Methods: Synchronization and Observation

基本的<em&gt;线虫</em&gt;方法：同步和观察

Research into the molecular and developmental biology of the nematode Caenorhabditis elegans was begun in the early seventies by Sydney Brenner and it has ...

Prostaglandin Extraction and Analysis in Caenorhabditis elegans

Prostaglandin Extraction and Analysis in Caenorhabditis elegans

前列腺素的提取与分析<em&gt;线虫</em

Caenorhabditis elegans is emerging as a powerful animal model to study the biology of lipids1-9. Prostaglandins are an important class of eicosanoids, ...

Measurement of mRNA Decay Rates in Saccharomyces cerevisiae Using rpb1-1 Strains

Measurement of mRNA Decay Rates in Saccharomyces cerevisiae Using rpb1-1 Strains

mRNA的衰变率的测量<em&gt;酿酒酵母</em&gt;使用<em&gt; rpb1-1</em&gt;株

mRNA steady state levels vary depending on environmental conditions. Regulation of the steady state accumulation levels of an mRNA ensures that the ...

Rodent Working Heart Model for the Study of Myocardial Performance and Oxygen Consumption

灭鼠工作心脏模型心肌的性能和氧耗的研究

Isolated working heart models have been used to understand the effects of loading conditions, heart rate and medications on myocardial performance in ways ...

Cultivation of Caenorhabditis elegans in Three Dimensions in the Laboratory

Cultivation of Caenorhabditis elegans in Three Dimensions in the Laboratory

栽培<em&gt;秀丽隐杆线虫</em&gt;在三个维度实验室

The use of genetic model organisms such as Caenorhabditis elegans has led to seminal discoveries in biology over the last five decades. Most of what we ...

Quantifying Tissue-Specific Proteostatic Decline in Caenorhabditis elegans

Quantifying Tissue-Specific Proteostatic Decline in Caenorhabditis elegans

量化秀丽隐杆线虫中组织特异性蛋白静剂下降

The ability to maintain proper function and folding of the proteome (protein homeostasis) declines during normal aging, facilitating the onset of a ...

Assessment of de novo Protein Synthesis Rates in Caenorhabditis elegans

Assessment of de novo Protein Synthesis Rates in Caenorhabditis elegans

卡诺哈布迪蒂斯 伊利根斯新蛋白合成率评估

Maintaining a healthy proteome is essential for cell and organismal homeostasis. Perturbation of the balance between protein translational control and ...