Name: plate-based large-scale cultivation of caenorhabditis elegans: sample preparation for the study of metabolic alterations in diabetes
Uploaded: 2018-08-24T14:00:00
Description: Watch this Scientific Journal Video about Plate-based Large-scale Cultivation of Caenorhabditis elegans: Sample Preparation for the Study of Metabolic Alterations in Diabetes at JoVE.com

这项研究得到德意志 Forschungsgemeinschaft (DFG) 在 IRTG 1874 "糖尿病微血管并发症" 和 CRC 1118 "反应性代谢物作为糖尿病晚期并发症的原因" 的支持。N2 和 CL2166 的线虫菌株由 CGC 提供, 由 NIH 研究基础设施项目办公室 (P40 OD010440) 资助。

注: 本协议分为五节。在1–3节中, 提出了大规模养殖C 型线虫的主要协议。4和5节为评估糖尿病代谢产物中出现的代谢产物提供了附加的协议。详细来说, 第1节描述了一个普通的大范围的板块文化。2节重点介绍了大量的C 线虫的转移, 而3节则解释了大规模样品的收获。4节解释了从样品中分离出的蛋白质, 5 节描述了随后的液相色谱-串联质谱 (LC-ms) 分析的样品制备方法。
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J., 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=Quantitative+screening+of+advanced+glycation+endproducts+in+cellular+and+extracellular+proteins+by+tandem+mass+spectrometry.">Quantitative screening of advanced glycation endproducts in cellular and extracellular proteins by tandem mass spectrometry.</a> Biochemical Journal. 375 (Pt 3), 581-592 (2003).</li><li>Karachalias, N., Babaei-Jadidi, R., Ahmed, N., Thornalley, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Accumulation+of+fructosyllysine+and+advanced+glycation+end+products+in+the+kidney,+retina+and+peripheral+nerve+of+streptozotocin-induced+diabetic+rats.">Accumulation of fructosyllysine and advanced glycation end products in the kidney, retina and peripheral nerve of streptozotocin-induced diabetic rats.</a> Biochemical Society Transactions. 31, 1423-1425 (2003).</li><li>Morcos, M., 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=Glyoxalase-1+prevents+mitochondrial+protein+modification+and+enhances+lifespan+in+Caenorhabditis+elegans.">Glyoxalase-1 prevents mitochondrial protein modification and enhances lifespan in Caenorhabditis elegans.</a> Aging Cell. 7 (2), 260-269 (2008).</li><li>Porta-de-la-Riva, M., Fontrodona, L., Villanueva, A., Cer&#243;n, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Basic+Caenorhabditis+elegans+methods:+synchronization+and+observation.">Basic Caenorhabditis elegans methods: synchronization and observation.</a> Journal of Visualized Experiments. 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该协议为大型线虫的大规模培养提供了可靠的途径, 获得了定量的结果。文献的结果可以复制, 如代表性结果所示。尽管这个用于收集大型线虫样本的协议似乎是一种直截了当的方法, 但还是有一定的缺陷需要考虑。关于线虫种群的同步问题, 本议定书描述了一种方法, 即用次氯酸钠和氢氧化钠漂白种群, 以破坏线虫, 并完全收获卵子。必须考虑到这种方法可能不适合所有实...

蛋白质分析, 如西方的印迹或质谱, 需要蛋白质的毫克。这种产量需要大规模培养数以百计的线虫, 这可以通过液体培养或固体介质通过洗涤转移线虫。流体和剪切应力诱导上皮钠通道 (ENaC) 的表达, 通过增加钠的吸收增加渗透应力, 可能改变线虫的寿命, 影响代谢分析1.因此, 该协议中的一些关键步骤, 以板块为基础的方法采取减少应力影响实验的变异性考虑。另一方面, 液体培养影响了线虫的表型, 复杂了2线虫的培养和准确数量的收集。此外, 活性物质可以改变的媒介成分, 并可能分布不均匀, 然后到达线虫。关于液体培养的局限性, 本议定书提供了一种新的方法来培养大规模的线虫样本。
线虫是一个模型有机体, 具有独特的网络302神经细胞, 占其所有细胞的 1/33。自引入科学以来, 许多同源和同源基因被描述, 放大它作为医学研究模型的价值。最近, 糖尿病视网膜病变 (前血管损伤) 的神经功能损害的证据已经提出4。线虫缺乏血管, 但包含一个独特的神经网络, 这使得它成为一个合适的模型来调查神经元的变化, 除了血管的。因此, 对于研究糖尿病并发症的一般机制,线虫有特殊的兴趣。糖尿病并发症的生化变化涉及年龄的形成, 这进一步影响了 ROS 的形成, 以应对高血糖5。年龄被发现在C. 线虫和贡献神经损伤6。慢性疾病通常是由复杂的多基因过程引起的, 需要多参数方法来评估其基本机制, 这里举例说明糖尿病并发症的评估。该协议可用于同时获取多个参数, 以及随后使用。通过省略液体与固体培养基之间的形态和代谢差异, 可以提高多参数方法的可比性和重现度。

<table>
	<tbody>
		<tr>
			<td>E. coli OP50</td>
			<td>CGC</td>
			<td>n/a</td>
			<td></td>
		</tr>
		<tr>
			<td>C. elegans N2</td>
			<td>CGC</td>
			<td>n/a</td>
			<td></td>
		</tr>
		<tr>
			<td>C. elegans CL2166</td>
			<td>CGC</td>
			<td>n/a</td>
			<td></td>
		</tr>
		<tr>
			<td>Petri dish, 60 x 15 mm</td>
			<td>Greiner One</td>
			<td>628161</td>
			<td></td>
		</tr>
		<tr>
			<td>Volumetric pipet, glas, 10 mL</td>
			<td>Neolab</td>
			<td>E-0413</td>
			<td></td>
		</tr>
		<tr>
			<td>Proteinase inhibitor cocktail tablets</td>
			<td>Roche</td>
			<td>04693124001</td>
			<td></td>
		</tr>
		<tr>
			<td>Non-denaturing lysate buffer:</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Tris-HCl, pH 8</td>
			<td>Sigma</td>
			<td>T3253</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodiumchloride (NaCl)</td>
			<td>Sigma</td>
			<td>S7653</td>
			<td></td>
		</tr>
		<tr>
			<td>Triton X-100</td>
			<td>Sigma</td>
			<td>X-100</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethylenediaminetetraacetic acid (EDTA)</td>
			<td>Sigma</td>
			<td>E5391</td>
			<td></td>
		</tr>
		<tr>
			<td>96-well plates, transparent bottom</td>
			<td>Brand</td>
			<td>781611</td>
			<td></td>
		</tr>
		<tr>
			<td>Infinite M200, plate reader</td>
			<td>Tecan</td>
			<td>30017581</td>
			<td></td>
		</tr>
		<tr>
			<td>Zirconium Oxide Beads, 0.5 mm</td>
			<td>Next advance</td>
			<td>ZROB05-RNA</td>
			<td></td>
		</tr>
		<tr>
			<td>Bullet Blender, homogenizer</td>
			<td>Next advance</td>
			<td>BBX24</td>
			<td></td>
		</tr>
		<tr>
			<td>Pepsin from porcine gastric mucosa</td>
			<td>Sigma</td>
			<td>P6887</td>
			<td></td>
		</tr>
		<tr>
			<td>Thymol</td>
			<td>Sigma</td>
			<td>T0501</td>
			<td></td>
		</tr>
		<tr>
			<td>Pronase E/ Protease from Streptomyces griseus </td>
			<td>Sigma</td>
			<td>P6911</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin-Streptomycin solution</td>
			<td>Sigma</td>
			<td>P43339</td>
			<td></td>
		</tr>
		<tr>
			<td>Prolidase from Porcine Kidney</td>
			<td>Sigma</td>
			<td>P6675</td>
			<td></td>
		</tr>
		<tr>
			<td>Aminopeptidase from Aeromonas proteolytica</td>
			<td>Sigma</td>
			<td>A8200</td>
			<td></td>
		</tr>
		<tr>
			<td>Amicon Ultra-0.5 Centrifugal Filter Unit</td>
			<td>Merckmillipore</td>
			<td>UFC501096</td>
			<td></td>
		</tr>
		<tr>
			<td>Basic Materials for plate culture are described in Reference 6.</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

plate-based large-scale cultivation of caenorhabditis elegans: sample preparation for the study of metabolic alterations in diabetes

在琼脂盘子上以大规模的方式培养秀丽线虫(线虫) 是费时费力的。该协议描述了一个简单而廉价的方法, 以获得大量的动物, 以分离蛋白质, 进行了西方印迹, 质谱, 或进一步蛋白质组学分析。此外, 在相同的培养条件下, immunostainings 线虫数量的增加和多种分析的整合也很容易实现。另外, 在不同实验条件下的板材之间的转移是方便的。在板块文化中常见的技术是用一根铂丝和用手术刀转移填充的琼脂块来转移单一的线虫.然而, 随着线虫数量的增加, 这些技术变得过于耗时。该协议描述了大规模的C 线虫文化, 包括许多步骤, 以尽量减少样本准备对蠕虫的生理影响。液体和剪切应力可以改变线虫的寿命和代谢过程, 因此需要详细描述关键步骤, 以检索可靠和可重现的结果。线虫是一种模型有机体, 由神经细胞组成, 可达 1/3, 但缺乏血管, 从而提供了单独研究独立于血管控制的神经元改变的可能性。最近, 在血管改变之前发现早期变性糖尿病视网膜病变。因此, 对于研究糖尿病并发症的一般机制,线虫有特殊的兴趣。例如, 在重现性中发现了晚期糖基化终产物 (年龄) 和活性氧 (ROS) 的增加形成. 通过对糖尿病引起的生化变化的研究, 本文介绍了处理足够大小样本以进行更广泛调查的协议。一般而言, 此协议对于需要大的C 线虫数和液体培养不合适的研究是有用的。

这里给出了创建大规模的C. 线虫文化在糖尿病研究中应用的例子。将参数与单个动物进行关联, 而不是将其规范化为总的蛋白质浓度是很有意义的。在需要少量线虫的化验中, 这可以很容易地通过计数线虫来完成。对于一个大规模的C. 线虫文化, 涉及数以百计的线虫每实验组, 这种方法是不方便的。在图 2中, 显示了线虫数量与蛋白?...

Watch this Scientific Journal Video about Plate-based Large-scale Cultivation of Caenorhabditis elegans: Sample Preparation for the Study of Metabolic Alterations in Diabetes at JoVE.com

Plate-based Large-scale Cultivation of Caenorhabditis elegans: Sample Preparation for the Study of Metabolic Alterations in Diabetes

本协议描述了一种在固体培养基上大规模培养秀丽线虫的方法。作为液体培养的替代品, 该协议允许在板基培养条件下获得不同尺度的参数。这增加了结果的可比性通过省略液体和固体媒介文化之间的形态学和新陈代谢的区别。

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Plate-based Large-scale Cultivation of Caenorhabditis elegans: Sample Preparation for the Study of Metabolic Alterations in Diabetes

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

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In contrast to aerobic organisms, strictly anaerobic microorganisms require the absence of oxygen and usually a low redox potential to initiate growth. As ...

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Fast oxidation of proteins (FPOP) is a hydroxyl radical protein footprinting (HRPF) method used to study protein structure, protein-ligand interactions, ...

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VMXm 光束线微晶样品制备管道

The mounting of microcrystals (&#60;10 &#181;m) for single crystal cryo-crystallography presents a non-trivial challenge. Improvements in data quality ...

Isolation of Active Caenorhabditis elegans Nuclear Extract and Reconstitution for In Vitro Transcription

Isolation of Active Caenorhabditis elegans Nuclear Extract and Reconstitution for In Vitro Transcription

活性 秀丽隐杆线 虫核提取物的分离及重建用于 体外 转录

Caenorhabditis elegans has been an important model system for biological research since it was introduced in 1963. However, C. elegans has not been fully ...

Large-Scale Multi-Omics Genome-Wide Association Studies (Mo-GWAS): Guidelines for Sample Preparation and Normalization

Large-Scale Multi-Omics Genome-Wide Association Studies Mo-GWAS: Guidelines for Sample Preparation and Normalization

大规模多组学全基因组关联研究 （Mo-GWAS）：样品制备和归一化指南

Both gas chromatography-mass spectrometry (GC-MS) and liquid chromatography-mass spectrometry (LC-MS) are widely used metabolomics approaches to detect ...