我们要感谢秀丽遗传学中心的菌株, 自然科学和工程研究理事会 (NSERC) 和加拿大创新基金会 (CFI) 供资。我们要感谢关丽珍博士 (细胞系统和解剖学部, UT 健康圣安东尼奥) 允许不受限制地使用她的实验室设施为所有C. 线虫实验和 Exing 博士 (副主任, 光学成像设备, UT 健康圣安东尼奥) 为协助与共焦显微术。此外, 我们亦多谢何议员提供支持和鼓励, 以协助拍摄影片。

1. 染色和图像肠道溶酶体
<ol><li>OP50 种子线虫生长培养基 (NGM) 板
	<ol>
<li>根据建议的协议19准备 NGM 板, 并允许在室温下将密封板干燥2天。</li>
		<li>将 OP50 细菌接种到无菌 Luria 汤 (磅) 汤中, 在37摄氏度或36小时的水浴中生长, 或直到外径介于0.2 和0.4 之间。避免使用细菌培养 > 1 或 od < 0.2。</li>
		<li>一旦 NGM 板干燥, 将一滴 (~ 30 µL) 的 OP50 接种到板的中心, 然后把下降到一个补丁大致是2厘米 x 2 厘米的大小。 注: 任何剩余的 OP50 接种可以储存在4摄氏度, 长达一个月。</li>
		<li>在接种干燥后反转 OP50 板 (大约10或15分钟), 然后将37摄氏度的板材孵化为36小时。</li>
		<li>36小时后, 从孵化箱中取出盘子, 储存在4摄氏度, 直到以后使用。 注: 板材应在4摄氏度的1月内良好。</li>
	</ol></li>
	<li>补充 cDCFDA 到 OP50
	<ol>
<li>在 OP50 板上补充 cDCFDA, 在二甲基亚砜 (亚砜) 上制备10毫米 cDCFDA 的工作库存。保持 cDCFDA 解决方案免受光照和存储在-20 摄氏度后使用。</li>
		<li>轻轻地将100µL 10 毫米 cDCFDA 溶液放到2厘米 x 2 厘米 OP50 贴片的表面, 这样整个贴片都被 cDCFDA 均匀覆盖。 注意: 这是非常重要的, 整个补丁的 OP50 覆盖 cDCFDA。如果需要, 使用多达150µL 的 cDCFDA 为每个板块。cDCFDA 解决方案也必须不超出 OP50 补丁的边界范围, 因为从 OP50 修补程序流出的任何 cDCFDA 不会被纳入食物中, 而且会导致染色强度降低。</li>
		<li>将 OP50 板放在工作台顶部的一个黑盒子里, 直到所有的 cDCFDA 溶液都被吸收到细菌斑块 (通常约25到30分钟)。</li>
	</ol></li>
	<li>使用 cDCFDA 染色蠕虫
	<ol>
<li>一旦 cDCFDA 完全渗透到 OP50 补丁, 每板放置不超过20蠕虫和孵化的板块反转在20°c 至少14小时。 注意: 建议将 cDCFDA 板放在不透明的盒子中, 以尽量减少光的照射。</li>
		<li>为了控制实验之间的摄入差异和规范 cDCFDA 信号, (推荐) 与 3-{2-[(1 H) 的 co 染色, 1 ' H-22 '-bipyrrol 5-基 kappaN (1)) methylidene]-2 H-咯-5-基 kappaN} N [2-(二甲基氨基) 乙] propanamidato) (difluoro) 硼 (2.5 µL 的新鲜制成的1毫米溶液), acidotropic 弱胺染料, 其荧光主要是非变异的酸性 pH 谱 (参见材料表的常用试剂名称)。 注意: 不要将蠕虫染色少于14小时, 因为这可能会提供不充分的 cDCFDA 染色强度, 并对溶酶体 pH 值作假解释。</li>
	</ol></li>
	<li>准备用于显微镜的幻灯片
	<ol>
<li>将两条带标签的磁带贴在平滑的平坦表面上, 如镜像 (图 1), 其间隔约为3英寸。</li>
		<li>将镜面表面涂上防水喷雾剂, 并擦拭多余液体。</li>
		<li>熔融2% 琼脂糖 (溶解在蒸馏 H2O), 直到完全液化, 然后在两条胶带之间放置一个50µL 滴琼脂糖, 用显微镜滑动迅速覆盖下降, 这样, 幻灯片垂直于带磁带。在琼脂糖凝固后, 在滑块下形成一个圆形垫, 翻转滑块, 将10到15µL 的5mM 钠叠氮化物 (NaN3) 添加到琼脂糖垫上。 注意: NaN3是一种细胞色素 C 抑制剂, 当用于低浓度时, 可逆 anesthetizes 蠕虫, 以便它们在成像过程中不会移动。</li>
		<li>从 OP50 板中提取蠕虫补充 cDCFDA, 并将其转移到一个干净的 NGM 板没有任何 OP50。允许蠕虫移动大约几秒钟, 允许大部分 OP50 从蠕虫表面移除, 然后将蠕虫转移到含有5毫米叠氮化钠的琼脂糖垫上。</li>
		<li>立即用盖子滑盖琼脂糖垫。一定要轻轻地放置在不施加太多的压力, 因为这可能导致蠕虫爆裂的封面滑动。 注: OP50 细菌辅以 cDCFDA 荧光显微镜下可视化, 因此, 重要的是要尽可能最好地清洁蠕虫之前, 把它们放在含有叠氮化钠的琼脂糖垫。如果准备6多株线虫(包括 N2、野生型控制), 最好在6批次中制备样品, 以确保蠕虫不会在琼脂糖垫上干燥。在某些菌株中, 由于来自盖板的压力, 外阴在延长的时间内开始破裂, 因此有必要进行相应的计划。</li>
	</ol></li>
	<li>共焦显微术 注: 某些显微镜具有内置功能, 可自动增加荧光强度, 以补偿可变水平的荧光。这种显微镜对 cDCFDA 染色的蠕虫可能无法正常工作, 因为它们本身就会增加样品的荧光强度。<ol>
<li>在标准共焦显微镜上进行成像, 使用激发/发射波长设置为 cDCFDA 488/530 nm。采用单平面图像 (不是 Z 栈) 的肠道溶酶体, 只使用集中 (最大信号强度) 的溶酶体进行强度量化。 注: 也许是由于染料的可用性不同, 肠细胞溶酶体直接后咽后保持 cDCFDA 染色强度, 无论基因型或年龄, 因此应注意避免这些细胞的成像溶酶体。</li>
		<li>图像的幻灯片包含2天老野生类型 N2 蠕虫首先。在这样做的同时, 调整共焦成像参数, 如激光功率, 针孔, 光圈, 以确保 cDCFDA 染色强度不饱和。 注意: 设置了这些设置后, 不要在当天的整个映像期间更改它们。</li>
		<li>使用60X 放大透镜捕获单个小肠平面的 1024 x 1024 像素图像 (每个蠕虫的3到4个图像)。 注: 蠕虫的 cDCFDA 发射光谱将产生一个单一的突出峰值在520毫微米与它报告的荧光光谱20, 提供一个特定的信号读数, 有别于肠道自发荧光 (图 3)。</li>
		<li>将原始共焦图像 (. lsm 文件) 导出为每个通道的. tiff 文件。</li>
		<li>接下来, 打开 ImageJ, 然后单击文件 |打开以打开要量化的图像文件。图像加载后, 使用 ImageJ 中的 "椭圆形状选择" 工具选择感兴趣的区域 (ROI)。</li>
		<li>此后, 单击分析 |测量以量化该 ROI 的荧光强度。选择第4-5 不同的聚焦溶酶每个图像和计算平均相对荧光强度每个区域的利益 (ROI)。</li>
		<li>对于每个图像, 测量溶酶体之间肠道区域的背景荧光强度。从溶酶体荧光强度值中减去背景荧光强度值。</li>
		<li>总计, 收集大约30到50个单独规范化的价值为 cDCFDA 强度 (6 到10动物) 为每株被测试。使用这些值绘制一个框和晶须图, 使用任何适当的统计软件。 注: 染色可能因温度、潜伏期和动物整体健康/年龄等因素的不同而有很大差异, 因此荧光强度比较仅在样品过程中与同一染色试验有关。因此, 在每个染色实验中处理控制是很重要的。使用任何适当的统计软件计算统计差异 (t测试)。</li>
		<li>图像的所有菌株从同一实验 (当天染色) 顺序, 注意不要改变任何成像参数。</li>
	</ol></li>
</ol>

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作者声明没有利益冲突。

各种细胞和分子事件导致衰老, 受生命史特征和遗传因素的影响。我们最近的研究22表明, 生殖周期通过调节溶酶体 pH 动态, 在控制躯体健康方面起着重要作用。我们表明, 溶酶介导的蛋白质水解是促进, 而动物积极繁殖的 v-atp 酶转录上调, 这反过来确保酸性溶酶。繁殖结束后, v-atp 酶表达下降, 溶酶体 alkalinize, 蛋白质聚集积累在这些细胞。
损害溶酶体酸化的?...

蛋白质团聚体的出现被广泛认为是真核细胞老化的标志1,2,3, 和其形成是细胞衰老的原则驱动因素4,5,6,7. 越来越多的证据表明, 随着细胞老化, 蛋白质分解代谢被削弱, 导致蛋白质聚集的增加。衰老细胞蛋白质水解的崩溃涉及自噬8的损伤以及蛋白酶介导的蛋白质降解9。最后, 在旧细胞中增加了不可逆转的蛋白质氧化, 进一步损害了蛋白质的分解代谢10。
自噬最初被认为是一个非选择性的过程, 以大量降解受损的蛋白质, 但最近的研究表明, 自噬是高度选择性的分解代谢的蛋白质聚集和功能失调的细胞器, 不可通过其他蛋白质清除机制11进行降解。在自噬过程中, 受损和聚集的蛋白质被隔离成一个叫做 autophagosome 的双膜囊泡。这个 autophagosome 然后与称为溶酶体的酸性细胞器融合, 导致 autophagosome 货物的降解12。溶酶体代表自噬性通路的终点, 参与不同的细胞过程, 如膜修复、转录控制和养分传感;突出它们在细胞稳态中的集中作用 (在 ref 13 中复习)。一些研究表明, 年龄依赖性的溶酶体功能下降与各种神经退行性疾病的相关性为13。一贯地, 在旧的细胞中恢复溶酶函数可以延缓衰老相关表型的出现,14,15。对 intralumen 环境组成的研究表明, 早期细胞溶酶酶功能的崩溃不是由于溶酶酶蛋白酶16的产生而减少的。另外, 有人建议, intralysosomal 酸度的损失, 它的酶活性的一个关键要求, 可能是在溶酶介导的蛋白质降解17的下降。为了能够探索这一假说, 必须开发试剂和协议, 以一种可复制和一致的方式来探测活细胞中溶酶体 pH 值的动态变化。
线虫的肠道是蠕虫的主要代谢组织, 是系统性稳态和寿命的重要调节因子。我们已经开发了测定蠕虫肠道溶酶体的酸性变化的检测方法, 以确定溶酶介导的蛋白质水解如何导致衰老。虽然以前在线虫中使用了 pH 敏感的显影标记肠道溶酶体, 但还没有努力建立一个成功的协议, 可以检测溶酶体 pH体内的小增加18.在这里, 我们提供了一种协议, 可用于检测C. 线虫肠道细胞中溶酶体酸度的损失, 使用一种简单而方便的喂养协议, 将 pH 敏感的荧光 (cDCFDA) 纳入 OP50 食品中。

<table><tbody><tr><td>OP50 (&lt;em&gt;E. coli&lt;/em&gt;)</td><td>Caenorhabditis Genetics Center</td><td></td><td>Order online at https://cgc.umn.edu/strain/OP50</td></tr><tr><td>5(6)-carboxy-2&amp;rsquo;,7&amp;rsquo;-dichlorofluorescein diacetate&amp;nbsp;</td><td>ThermoFisher</td><td>C369</td><td>Commonly known as cDCFDA</td></tr><tr><td>9-diethylamino-5H-benzo(a)phenoxazine-5-one and (3-{2-[(1H,1&#039;H-2,2&#039;-bipyrrol-5-yl-kappaN(1))methylidene]-2H-pyrrol-5-yl-kappaN}-N-[2-(dimethylamino)ethyl]propanamidato)(difluoro)boron</td><td>ThermoFisher</td><td>L7528</td><td>Commonly known as Lysotracker Red</td></tr><tr><td>Confocal microscope (e.g. Zeiss LSM 510)</td><td></td><td></td><td></td></tr><tr><td>ImageJ</td><td></td><td></td><td>Download for free from https://imagej.nih.gov/ij/download.html</td></tr><tr><td>LB Broth powder</td><td>ThermoFisher</td><td>22700041</td><td></td></tr><tr><td>Bacto Agar</td><td>Sigma</td><td>A5306-1KG</td><td></td></tr><tr><td>NaCl</td><td>Sigma</td><td>S9888</td><td></td></tr><tr><td>Bacto Peptone</td><td>Fisher Scientific</td><td>S71604</td><td></td></tr><tr><td>Cholesterol powder</td><td>Sigma</td><td>C3045&amp;nbsp;</td><td></td></tr><tr><td>CaCl2</td><td>Sigma</td><td>449709</td><td></td></tr><tr><td>MgSO4</td><td>Sigma</td><td>M7506</td><td></td></tr><tr><td>K3PO4</td><td>Sigma</td><td>P5629</td><td></td></tr><tr><td>Sodium Azide</td><td>Sigma</td><td>S2002</td><td></td></tr><tr><td>DMSO</td><td>Sigma</td><td>D8418</td><td></td></tr><tr><td>Microscope Slides</td><td>VWR</td><td>48311-703</td><td></td></tr><tr><td>Cover Slips</td><td>ThermoFisher</td><td>3406</td><td></td></tr><tr><td>Agarose</td><td>Sigma</td><td>A6013</td><td></td></tr><tr><td>Incubator</td><td></td><td></td><td></td></tr><tr><td>Mirror or other smooth flat surface</td><td></td><td></td><td></td></tr></tbody></table>

assessing lysosomal alkalinization in the intestine of live caenorhabditis elegans

线虫秀丽线虫(C. 线虫) 是一种广泛用于研究长寿和发育途径的模型系统。这种研究通过动物的透明性、向前和反向基因检测的能力、产生荧光标记蛋白的相对容易性以及荧光染料的使用来促进, 它们可以杏仁到早期胚胎或纳入其食物 (大肠杆菌菌株 OP50) 标签细胞器 (例如9-二乙基-5 H 苯并 (a) phenoxazine-5-一和 (3-{2-[(1 H, 1 ' H-22 '-bipyrrol 5-基 kappaN (1)) methylidene]-2 H-咯-5-基 kappaN}-N-[2-(二甲基氨基) 乙基] propanamidato) (difluoro) 硼)。在这里, 我们提出使用荧光 pH 敏感染料, 染色肠道溶酶体, 提供了动态, 生理变化的溶酶体酸度在活蠕虫的视觉读数。该协议不测量溶酶体 pH 值, 而是旨在建立一个可靠的方法来评估溶酶酸度的生理相关变化。cDCFDA 是一种细胞 permeant 化合物, 转化为荧光荧光 5-(和-6) 羧基-2 ', 7 '-dichlorofluorescein (cDCF) 水解后, 细胞内酯。质子内溶酶体陷阱 cDCF 在这些细胞器中, 在那里积聚。该染料由于其4.8 的低碱性, 已被用作酵母中的 pH 传感器。在这里, 我们描述了使用 cDCFDA 作为食物补充, 以评估肠道溶酶体的酸度在线虫.这种技术允许检测活动物中的碱化溶酶体,并具有广泛的实验应用, 包括对衰老、自噬和溶酶合成的研究。

cDCFDA 渍溶酶在 ph 依赖性的方式, 它的低 pKa 和准备吸收入溶酶使它成为理想的 pH 传感器21。cDCFDA 染色强度与溶酶体 ph 值成反比 (即染色强度随 pH 值的降低而增加)18,22。cDCFDA 信号在动物溶酶体中一直很弱, 在20毫米的氯喹, 一个溶酶体酸化抑制剂, 和在蠕虫耗尽的 v-atp 酶, 蛋白质复合体的膜上的溶酶, 质子?...

Watch this Scientific Journal Video about Assessing Lysosomal Alkalinization in the Intestine of Live Caenorhabditis elegans at JoVE.com

Assessing Lysosomal Alkalinization in the Intestine of Live Caenorhabditis elegans

用 pH 敏感的生命染料 5 (6)-羧基-2 ', 7 '-dichlorofluorescein 双乙酸 (cDCFDA) 研究了在线虫的肠道中溶酶体酸度损失的分步指南

评估溶酶体碱化在活的秀丽线虫中的肠道

The nematode Caenorhabditis elegans (C. elegans) is a model system that is widely used to study longevity and developmental pathways. Such studies are ...

assessing-lysosomal-alkalinization-intestine-live-caenorhabditis

Universidad de Cartagena UDEC

Research

JoVE Journal

Developmental Biology

Assessing Lysosomal Alkalinization in the Intestine of Live Caenorhabditis elegans

5.9K 次观看.  University of Saskatchewan. 用 pH 敏感的生命染料 5 (6)-羧基-2 ', 7 '-dichlorofluorescein 双乙酸 (cDCFDA) 研究了在线虫的肠道中溶酶体酸度损失的分步指南

视频: Assessing Lysosomal Alkalinization in the Intestine of Live Caenorhabditis elegans

Small-Scale Colorimetric Assays of Intracellular Lactate and Pyruvate in the Nematode Caenorhabditis elegans

Lactate and pyruvate are key intermediates of intracellular energy metabolic pathways. Monitoring the lactate/pyruvate ratio in cells helps to determine whether there is an imbalance in age-related energy metabolism between mitochondrial oxidative phosphorylation and aerobic glycolysis. Here, we show the utilization of commercial colorimetric assay kits for lactate and pyruvate in the model organism C. elegans. Recently, the sensitivity and accuracy of the colorimetric/fluorimetric assay kits have been improved greatly by the research and development conducted by reagent manufacturers. The improved reagents have enabled the use of small-scale assays with a 96-well plate in C. elegans. In general, a fluorimetric assay is superior in sensitivity to a colorimetric assay; however, the colorimetric approach is more suitable for the use in common laboratories. Another important issue in these assays for quantitative determination is protein precipitation of homogenized C. elegans samples. In our protein precipitation method, common precipitants (e.g., trichloroacetic acid, perchloric acid and metaphosphoric acid) are used for sample preparation. A protein-free assay sample is prepared by directly adding cold precipitant (final concentration of 5%) during homogenization.

Small-Scale Colorimetric Assays of Intracellular Lactate and Pyruvate in the Nematode Caenorhabditis elegans

We describe a modified small-scale extraction and colorimetric assays of lactate and pyruvate in the nematode C. elegans. When utilizing commercial assay kits, the technical development of their sensitivity and accuracy is important. Protein precipitation in extraction is the most critical step for the quantitative determination of intracellular metabolites.

线虫线虫中乳酸和丙酮酸细胞的小尺度比色测定

Lactate and pyruvate are key intermediates of intracellular energy metabolic pathways. Monitoring the lactate/pyruvate ratio in cells helps to determine ...

科研

生物化学

The C. elegans Intestine As a Model for Intercellular Lumen Morphogenesis and In Vivo Polarized Membrane Biogenesis at the Single-cell Level: Labeling by Antibody Staining, RNAi Loss-of-function Analysis and Imaging

Multicellular tubes, fundamental units of all internal organs, are composed of polarized epithelial or endothelial cells, with apical membranes lining the lumen and basolateral membranes contacting each other and/or the extracellular matrix. How this distinctive membrane asymmetry is established and maintained during organ morphogenesis is still an unresolved question of cell biology. This protocol describes the C. elegans intestine as a&#160;model for the analysis of polarized membrane biogenesis during tube morphogenesis, with emphasis on apical membrane and lumen biogenesis. The C. elegans twenty-cell single-layered intestinal epithelium is arranged into a simple bilaterally symmetrical tube, permitting analysis on a single-cell level. Membrane polarization occurs concomitantly with polarized cell division and migration during early embryogenesis, but de novo polarized membrane biogenesis continues throughout larval growth, when cells no longer proliferate and move. The latter setting allows one to separate subcellular changes that simultaneously mediate these different polarizing processes, difficult to distinguish in most polarity models. Apical-, basolateral membrane-, junctional-, cytoskeletal- and endomembrane components can be labeled and tracked throughout development by GFP fusion proteins, or assessed by in situ antibody staining. Together with the organism&#39;s genetic versatility, the C. elegans intestine thus provides a unique in vivo model for the visual, developmental, and molecular genetic analysis of polarized membrane and tube biogenesis. The specific methods (all standard) described here include how to: label intestinal subcellular components by antibody staining; analyze genes involved in polarized membrane biogenesis by loss-of-function studies adapted to the typically essential tubulogenesis genes; assess polarity defects during different developmental stages; interpret phenotypes by epifluorescence, differential interference contrast (DIC) and confocal microscopy; quantify visual defects. This protocol can be adapted to analyze any of the often highly conserved molecules involved in epithelial polarity, membrane biogenesis, tube and lumen morphogenesis.

The C. elegans Intestine As a Model for Intercellular Lumen Morphogenesis and In Vivo Polarized Membrane Biogenesis at the Single-cell Level: Labeling by Antibody Staining, RNAi Loss-of-function Analy

The transparent C. elegans intestine can serve as an "in vivo tissue chamber" for studying apicobasal membrane and lumen biogenesis at the single-cell and subcellular level during multicellular tubulogenesis. This protocol describes how to combine standard labeling, loss-of-function genetic/RNAi and microscopic approaches to dissect these processes on a molecular level.

C. 线虫小肠作为细胞间腔形态发生和体内极化膜生物单细胞水平的模型: 抗体染色、rna 干扰功能分析和成像标记

Multicellular tubes, fundamental units of all internal organs, are composed of polarized epithelial or endothelial cells, with apical membranes lining the ...

发育生物学

The C. elegans Excretory Canal as a Model for Intracellular Lumen Morphogenesis and In Vivo Polarized Membrane Biogenesis in a Single Cell: labeling by GFP-fusions, RNAi Interaction Screen and Imaging

The four C. elegans excretory canals are narrow tubes extended through the length of the animal from a single cell, with almost equally far extended intracellular endotubes that build and stabilize the lumen with a membrane and submembraneous cytoskeleton of apical character. The excretory cell expands its length approximately 2,000 times to generate these canals, making this model unique for the in vivo assessment of de novo polarized membrane biogenesis, intracellular lumen morphogenesis and unicellular tubulogenesis. The protocol presented here shows how to combine standard labeling, gain- and loss-of-function genetic or RNA interference (RNAi)-, and microscopic approaches to use this model to visually dissect and functionally analyze these processes on a molecular level. As an example of a labeling approach, the protocol outlines the generation of transgenic animals with fluorescent fusion proteins for live analysis of tubulogenesis. As an example of a genetic approach, it highlights key points of a visual RNAi-based interaction screen designed to modify a gain-of-function cystic canal phenotype. The specific methods described are how to: label and visualize the canals by expressing fluorescent proteins; construct a targeted RNAi library and strategize RNAi screening for the molecular analysis of canal morphogenesis; visually assess modifications of canal phenotypes; score them by dissecting fluorescence microscopy; characterize subcellular canal components at higher resolution by confocal microscopy; and quantify visual parameters. The approach is useful for the investigator who is interested in taking advantage of the C. elegans excretory canal for identifying and characterizing genes involved in the phylogenetically conserved processes of intracellular lumen and unicellular tube morphogenesis.

The C. elegans Excretory Canal as a Model for Intracellular Lumen Morphogenesis and In Vivo Polarized Membrane Biogenesis in a Single Cell: labeling by GFP-fusions, RNAi Interaction Screen and Imaging

The C. elegans excretory canal is a unique single-cell model for the visual in vivo analysis of de novo polarized membrane biogenesis. This protocol describes a combination of standard genetic/RNAi and imaging approaches, adaptable for the identification and characterization of molecules directing unicellular tubulogenesis, and apical membrane and lumen biogenesis.

C. 线虫排泄管作为细胞内腔形态发生和体内极化膜生物在单个细胞中的模型: GFP-融合、rna 干扰界面和成像的标记

The four C. elegans excretory canals are narrow tubes extended through the length of the animal from a single cell, with almost equally far extended ...

Immobilization of Caenorhabditis elegans to Analyze Intracellular Transport in Neurons

Axonal transport and intraflagellar transport (IFT) are essential for axon and cilia morphogenesis and function. Kinesin superfamily proteins and dynein are molecular motors that regulate anterograde and retrograde transport, respectively. These motors use microtubule networks as rails. Caenorhabditis elegans (C. elegans) is a powerful model organism to study axonal transport and IFT in vivo. Here, I describe a protocol to observe axonal transport and IFT in living C. elegans. Transported cargo can be visualized by tagging cargo proteins using fluorescent proteins such as green fluorescent protein (GFP). C. elegans is transparent and GFP-tagged cargo proteins can be expressed in specific cells under cell-specific promoters. Living worms can be fixed by microbeads on 10% agarose gel without killing or anesthetizing the worms. Under these conditions, cargo movement can be directly observed in the axons and cilia of living C. elegans without dissection. This method can be applied to the observation of any cargo molecule in any cells by modifying the target proteins and/or the cells they are expressed in. Most basic proteins such as molecular motors and adaptor proteins that are involved in axonal transport and IFT are conserved in C. elegans. Compared to other model organisms, mutants can be obtained and maintained more easily in C. elegans. Combining this method with various C. elegans mutants can clarify the molecular mechanisms of axonal transport and IFT.

Immobilization of Caenorhabditis elegans to Analyze Intracellular Transport in Neurons

Caenorhabditis elegans (C. elegans) is a good model to study axonal and intracellular transport. Here, I describe a protocol for in vivo recording and analysis of axonal and intraflagellar transport in C. elegans.

固定化的线虫线虫分析神经元内细胞的转运

Axonal transport and intraflagellar transport (IFT) are essential for axon and cilia morphogenesis and function. Kinesin superfamily proteins and dynein ...

A Caenorhabditis elegans Model System for Amylopathy Study

Amylopathy is a term that describes abnormal synthesis and accumulation of amyloid beta (A&beta;) in tissues with time. A&beta; is a hallmark of Alzheimer's disease (AD) and is found in Lewy body dementia, inclusion body myositis and cerebral amyloid angiopathy 1-4. Amylopathies progressively develop with time. For this reason simple organisms with short lifespans may help to elucidate molecular aspects of these conditions. Here, we describe experimental protocols to study A&beta;-mediated neurodegeneration using the worm Caenorhabditis elegans. Thus, we construct transgenic worms by injecting DNA encoding human A&beta;42 into the syncytial gonads of adult hermaphrodites. Transformant lines are stabilized by a mutagenesis-induced integration. Nematodes are age synchronized by collecting and seeding their eggs. The function of neurons expressing A&beta;42 is tested in opportune behavioral assays (chemotaxis assays). Primary neuronal cultures obtained from embryos are used to complement behavioral data and to test the neuroprotective effects of anti-apoptotic compounds.

A Caenorhabditis elegans Model System for Amylopathy Study

We describe methods to study aspects of amylopathies in the worm C. elegans. We show how to construct worms expressing human A&beta;42 in neurons and how to test their function in behavioral assays. We further show how to obtain primary neuronal cultures that can be used for pharmacological testing.

一<em&gt;线虫</em&gt;研究Amylopathy模型系统

Amylopathy is a term that describes abnormal synthesis and accumulation of amyloid beta (A&beta;) in tissues with time. A&beta; is a hallmark of ...

神经科学

Stable Isotopic Profiling of Intermediary Metabolic Flux in Developing and Adult Stage Caenorhabditis elegans

Stable isotopic profiling has long permitted sensitive investigations of the metabolic consequences of genetic mutations and/or pharmacologic therapies in cellular and mammalian models. Here, we describe detailed methods to perform stable isotopic profiling of intermediary metabolism and metabolic flux in the nematode, Caenorhabditis elegans. Methods are described for profiling whole worm free amino acids, labeled carbon dioxide, labeled organic acids, and labeled amino acids in animals exposed to stable isotopes either from early development on nematode growth media agar plates or beginning as young adults while exposed to various pharmacologic treatments in liquid culture. Free amino acids are quantified by high performance liquid chromatography (HPLC) in whole worm aliquots extracted in 4% perchloric acid. Universally labeled 13C-glucose or 1,6-13C2-glucose is utilized as the stable isotopic precursor whose labeled carbon is traced by mass spectrometry in carbon dioxide (both atmospheric and dissolved) as well as in metabolites indicative of flux through glycolysis, pyruvate metabolism, and the tricarboxylic acid cycle. Representative results are included to demonstrate effects of isotope exposure time, various bacterial clearing protocols, and alternative worm disruption methods in wild-type nematodes, as well as the relative extent of isotopic incorporation in mitochondrial complex III mutant worms (isp-1(qm150)) relative to wild-type worms. Application of stable isotopic profiling in living nematodes provides a novel capacity to investigate at the whole animal level real-time metabolic alterations that are caused by individual genetic disorders and/or pharmacologic therapies.

Stable Isotopic Profiling of Intermediary Metabolic Flux in Developing and Adult Stage Caenorhabditis elegans

Stable isotopic profiling by gas chromatography mass spectrometric analysis of intermediary metabolic flux is described in the nematode, Caenorhabditis elegans. Methods are detailed for assessing isotopic enrichment in carbon dioxide, organic acids, and amino acids following isotope exposure either during development on agar plates or during adulthood in liquid culture.

在开发和成年阶段的中介代谢通量的稳定同位素分析线虫</em

Stable isotopic profiling has long permitted sensitive investigations of the metabolic consequences of genetic mutations and/or pharmacologic therapies in ...

生物学

RNA Fluorescence in situ Hybridization (FISH) to Visualize Microbial Colonization and Infection in Caenorhabditis elegans Intestines

The intestines of wild Caenorhabditis nematodes are inhabited by a variety of microorganisms, including gut microbiome bacteria and pathogens, such as microsporidia and viruses. Because of the similarities between Caenorhabditis elegans and mammalian intestinal cells, as well as the power of the C. elegans system, this host has emerged as a model system to study host intestine-microbe interactions in vivo. While it is possible to observe some aspects of these interactions with bright-field microscopy, it is difficult to accurately classify microbes and characterize the extent of colonization or infection without more precise tools. RNA fluorescence in situ hybridization (FISH) can be used as a tool to identify and visualize microbes in nematodes from the wild or to experimentally characterize and quantify infection in nematodes infected with microbes in the lab. FISH probes, labeling the highly abundant small subunit ribosomal RNA, produce a bright signal for bacteria and microsporidian cells. Probes designed to target conserved regions of ribosomal RNA common to many species can detect a broad range of microbes, whereas targeting divergent regions of the ribosomal RNA is useful for narrower detection. Similarly, probes can be designed to label viral RNA. A protocol for RNA FISH staining with either paraformaldehyde (PFA) or acetone fixation is presented. PFA fixation is ideal for nematodes associated with bacteria, microsporidia, and viruses, whereas acetone fixation is necessary for the visualization of microsporida spores. Animals were first washed and fixed in paraformaldehyde or acetone. After fixation, FISH probes were incubated with samples to allow for the hybridization of probes to the desired target. The animals were again washed and then examined on microscope slides or using automated approaches. Overall, this FISH protocol enables detection, identification, and quantification of the microbes that inhabit the C. elegans intestine, including microbes for which there are no genetic tools available.

RNA Fluorescence in situ Hybridization FISH to Visualize Microbial Colonization and Infection in Caenorhabditis elegans Intestines

Intestinal microbes, including extracellular bacteria and intracellular pathogens like the Orsay virus and microsporidia (fungi), are often associated with wild Caenorhabditis nematodes. This article presents a protocol for detecting and quantifying microbes that colonize and/or infect C. elegans nematodes, and for measuring pathogen load after controlled infections in the lab.

RNA荧光 原位 杂交（FISH）可视化 秀丽隐杆线虫 肠道中的微生物定植和感染

The intestines of wild Caenorhabditis nematodes are inhabited by a variety of microorganisms, including gut microbiome bacteria and pathogens, such as ...

免疫与感染

An Anoxia-starvation Model for Ischemia/Reperfusion in C. elegans

Protocols for anoxia/starvation in the genetic model organism C. elegans simulate ischemia/reperfusion. Worms are separated from bacterial food and placed under anoxia for 20 hr&#160;(simulated ischemia), and subsequently moved to a normal atmosphere with food (simulated reperfusion). This experimental paradigm results in increased death and neuronal damage, and techniques are presented to assess organism viability, alterations to the morphology of touch neuron processes, as well as touch sensitivity, which represents the behavioral output of neuronal function. Finally, a method for constructing hypoxic incubators using common kitchen storage containers is described. The addition of a mass flow control unit allows for alterations to be made to the gas mixture in the custom incubators, and a circulating water bath allows for both temperature control and makes it easy to identify leaks. This method provides a low cost alternative to commercially available units.

An Anoxia-starvation Model for Ischemia/Reperfusion in C. elegans

A protocol is described that uses anoxia/starvation in C. elegans to model ischemia/reperfusion. Functional outcomes include increased mortality, visible abnormalities in GFP-labeled neuronal processes, and impaired behavioral responses that require neuronal function.

对缺血/再灌注在缺氧，饥饿型号<em&gt; C。线虫</em

Protocols for anoxia/starvation in the genetic model organism C. elegans simulate ischemia/reperfusion. Worms are separated from bacterial food and placed ...

Measuring the Effects of Bacteria and Chemicals on the Intestinal Permeability of Caenorhabditis elegans

In living organisms, intestinal hyperpermeability is a serious symptom that leads to many inflammatory bowel diseases (IBDs). Caenorhabditis elegans is a nonmammalian animal model that is widely used as an assay system due to its short lifespan, transparency, cost-effectiveness, and lack of animal ethics issues. In this study, a method was developed to investigate the effects of different bacteria and 3,3'-diindolylmethane (DIM) on the intestinal permeability of C. elegans with a high-throughput image analysis system. The worms were infected with different gut bacteria or cotreated with DIM for 48 h and fed with fluorescein isothiocyanate (FITC)-dextran overnight. Then, the intestinal permeability was examined by comparing the fluorescence images and the fluorescence intensity inside the worm bodies. This method may also have the potential to identify probiotic and pathogenic intestinal bacteria that affect intestinal permeability in the animal model and is effective for examining the effects of harmful or health-promoting chemicals on intestinal permeability and intestinal health. However, this protocol also has some considerable limitations at the genetic level, especially for determining which genes are altered to control illness, because this method is mostly used for phenotypic determination. In addition, this method is limited to determining exactly which pathogenic substrates cause inflammation or increase the permeability of the worms' intestines during infection. Therefore, further in-depth studies, including investigation of the molecular genetic mechanism using mutant bacteria and nematodes as well as chemical component analysis of bacteria, are required to fully evaluate the function of bacteria and chemicals in determining intestinal permeability.

Measuring the Effects of Bacteria and Chemicals on the Intestinal Permeability of Caenorhabditis elegans

This protocol describes how to measure intestinal permeability of Caenorhabditis elegans. This method is helpful for basic biological research on intestinal health related to the interaction between intestinal bacteria and their host and for screening to identify probiotic and chemical agents to cure leaky gut syndrome and inflammatory bowel diseases.

测定细菌和化学物质对开牛结不二病的肠道渗透性的影响

In living organisms, intestinal hyperpermeability is a serious symptom that leads to many inflammatory bowel diseases (IBDs). Caenorhabditis elegans is a ...

医学

Hand Dissection of Caenorhabditis elegans Intestines

Comprised of only 20 cells, the Caenorhabditis elegans intestine&#160;is the nexus of many life-supporting functions, including digestion, metabolism, aging, immunity, and environmental response. Critical interactions between the C. elegans host and its environment converge within the intestine, where gut microbiota concentrate. Therefore, the ability to isolate intestine tissue away from the rest of the worm is necessary to assess intestine-specific processes. This protocol describes a method for hand dissecting adult C. elegans intestines. The procedure can be performed in fluorescently labeled strains for ease or training purposes. Once the technique is perfected, intestines can be collected from unlabeled worms of any genotype. This microdissection approach allows for the simultaneous capture of host intestinal tissue and gut microbiota, a benefit to many microbiome studies. As such, downstream applications for the intestinal preparations generated by this protocol can include but are not limited to RNA isolation from intestinal cells and DNA isolation from captured microbiota. Overall, hand dissection of C. elegans intestines affords a simple and robust method to investigate critical aspects of intestine biology.

Hand Dissection of Caenorhabditis elegans Intestines

The present protocol describes a procedure for isolating intestines from adult Caenorhabditis elegans nematode worms by hand for input in genomics, proteomics, microbiome, or other assays.

秀丽隐杆线虫肠的手部解剖

Comprised of only 20 cells, the Caenorhabditis elegans intestine&#160;is the nexus of many life-supporting functions, including digestion, metabolism, ...

评估溶酶体碱化在活的秀丽线虫中的肠道

摘要

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线虫线虫中乳酸和丙酮酸细胞的小尺度比色测定

C. 线虫小肠作为细胞间腔形态发生和体内极化膜生物单细胞水平的模型: 抗体染色、rna 干扰功能分析和成像标记

C. 线虫排泄管作为细胞内腔形态发生和体内极化膜生物在单个细胞中的模型: GFP-融合、rna 干扰界面和成像的标记

固定化的线虫线虫分析神经元内细胞的转运

一

在开发和成年阶段的中介代谢通量的稳定同位素分析线虫

RNA荧光原位杂交（FISH）可视化秀丽隐杆线虫肠道中的微生物定植和感染

对缺血/再灌注在缺氧，饥饿型号

测定细菌和化学物质对开牛结不二病的肠道渗透性的影响

秀丽隐杆线虫肠的手部解剖

评估溶酶体碱化在活的秀丽线虫中的肠道

摘要

探索更多视频

线虫线虫中乳酸和丙酮酸细胞的小尺度比色测定

C. 线虫小肠作为细胞间腔形态发生和体内极化膜生物单细胞水平的模型: 抗体染色、rna 干扰功能分析和成像标记

C. 线虫排泄管作为细胞内腔形态发生和体内极化膜生物在单个细胞中的模型: GFP-融合、rna 干扰界面和成像的标记

固定化的线虫线虫分析神经元内细胞的转运

一

在开发和成年阶段的中介代谢通量的稳定同位素分析线虫

RNA荧光 原位 杂交（FISH）可视化 秀丽隐杆线虫 肠道中的微生物定植和感染

对缺血/再灌注在缺氧，饥饿型号

测定细菌和化学物质对开牛结不二病的肠道渗透性的影响

秀丽隐杆线虫肠的手部解剖

RNA荧光原位杂交（FISH）可视化秀丽隐杆线虫肠道中的微生物定植和感染