Name: a high-throughput assay for the prediction of chemical toxicity by automated phenotypic profiling of caenorhabditis elegans
Uploaded: 2019-03-14T14:45:00
Description: Watch this Scientific Journal Video about A High-throughput Assay for the Prediction of Chemical Toxicity by Automated Phenotypic Profiling of Caenorhabditis elegans at JoVE.com

作者感谢 cgc 善意地发送了线虫。这项工作得到了中国国家重点研究与发展计划 (#2018YFC1603102、#2018YFC1602705) 的支持;国家自然科学基金授予 (#31401025, #81273108, #81641184), 北京首都健康研究与开发专项项目 (#2011-1013-03), 北京环境毒理学重点实验室开放基金 (#2015 hjdl03), 中国山东省自然科学基金 (zr2017bf041)。

该协议遵循了中国北京疾病预防控制中心动物伦理委员会的动物护理指南。
1. 化学制剂
<ol><li>获取化学品 (表 1和材料表)。</li>
	<li>确定单个化学品的最高和最低剂量, 对蠕虫的最低浓度为100% 杀伤力 (lc100, 24小时), 最大浓度为100% 非杀伤力 (lc0, 24小时)。使用至少6次浓度最高的稀释剂 (表 1)。 注: 进行初步蠕虫杀伤力测试9<...

<ol>
	<li>Anderson, G. L., 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=Assessing+behavioral+toxicity+with+Caenorhabditis+elegans.">Assessing behavioral toxicity with Caenorhabditis elegans.</a> Environmental Toxicology and Chemistry. 23 (5), 1235-1240 (2004).</li><li>Boyd, W. A., 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=A+high-throughput+method+for+assessing+chemical+toxicity+using+a+Caenorhabditis+elegans+reproduction+assay.">A high-throughput method for assessing chemical toxicity using a Caenorhabditis elegans reproduction assay.</a> Toxicology and Applied Pharmacology. 245 (2), 153-159 (2010).</li><li>Boyd, W. A., Williams, P. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+of+the+sensitivity+of+three+nematode+species+to+copper+and+their+utility+in+aquatic+and+soil+toxicity+tests.">Comparison of the sensitivity of three nematode species to copper and their utility in aquatic and soil toxicity tests.</a> Environmental Toxicology and Chemistry. 22 (11), 2768-2774 (2003).</li><li>Dengg, M., van Meel, 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=Caenorhabditis+elegans+as+model+system+for+rapid+toxicity+assessment+of+pharmaceutical+compounds.">Caenorhabditis elegans as model system for rapid toxicity assessment of pharmaceutical compounds.</a> Journal of Pharmacological and Toxicological Methods. 50 (3), 209-214 (2004).</li><li>Schouest, 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=Toxicological+assessment+of+chemicals+using+Caenorhabditis+elegans+and+optical+oxygen+respirometry.">Toxicological assessment of chemicals using Caenorhabditis elegans and optical oxygen respirometry.</a> Environmental Toxicology and Chemistry. 28 (4), 791-799 (2009).</li><li>Sprando, R. L., 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=A+method+to+rank+order+water+soluble+compounds+according+to+their+toxicity+using+Caenorhabditis+elegans,+a+Complex+Object+Parametric+Analyzer+and+Sorter,+and+axenic+liquid+media.">A method to rank order water soluble compounds according to their toxicity using Caenorhabditis elegans, a Complex Object Parametric Analyzer and Sorter, and axenic liquid media.</a> Food and Chemical Toxicology. 47 (4), 722-728 (2009).</li><li>Wang, D., Xing, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Assessment+of+locomotion+behavioral+defects+induced+by+acute+toxicity+from+heavy+metal+exposure+in+nematode+Caenorhabditis+elegans.">Assessment of locomotion behavioral defects induced by acute toxicity from heavy metal exposure in nematode Caenorhabditis elegans.</a> Journal of Environmental Sciences (China). 20 (9), 1132-1137 (2008).</li><li>Leung, M. C., 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=Caenorhabditis+elegans:+an+emerging+model+in+biomedical+and+environmental+toxicology.">Caenorhabditis elegans: an emerging model in biomedical and environmental toxicology.</a> Toxicological Sciences. 106 (1), 5-28 (2008).</li><li>Li, Y., 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=Correlation+of+chemical+acute+toxicity+between+the+nematode+and+the+rodent.">Correlation of chemical acute toxicity between the nematode and the rodent.</a> Toxicology Research. 2 (6), 403-412 (2013).</li><li>Boyd, W. A., 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=Effects+of+genetic+mutations+and+chemical+exposures+on+Caenorhabditis+elegans+feeding:+evaluation+of+a+novel,+high-throughput+screening+assay.">Effects of genetic mutations and chemical exposures on Caenorhabditis elegans feeding: evaluation of a novel, high-throughput screening assay.</a> PLoS One. 2 (12), 1259 (2007).</li><li>Xian, B., 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=WormFarm:+a+quantitative+control+and+measurement+device+toward+automated+Caenorhabditis+elegans+aging+analysis.">WormFarm: a quantitative control and measurement device toward automated Caenorhabditis elegans aging analysis.</a> Aging Cell. 12 (3), 398-409 (2013).</li><li>Gao, 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=Classification+and+prediction+of+toxicity+of+chemicals+using+an+automated+phenotypic+profiling+of+Caenorhabditis+elegans.">Classification and prediction of toxicity of chemicals using an automated phenotypic profiling of Caenorhabditis elegans.</a> BMC Pharmacology and Toxicology. 19 (1), 18 (2018).</li><li>Moyson, 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=Mixture+effects+of+copper,+cadmium,+and+zinc+on+mortality+and+behavior+of+Caenorhabditis+elegans.">Mixture effects of copper, cadmium, and zinc on mortality and behavior of Caenorhabditis elegans.</a> Environmental Toxicology and Chemistry. 37 (1), 145-159 (2018).</li><li>Wang, X., 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=Lifespan+extension+in+Caenorhabditis+elegans+by+DMSO+is+dependent+on+sir-2.1+and+daf-16.">Lifespan extension in Caenorhabditis elegans by DMSO is dependent on sir-2.1 and daf-16.</a> Biochemical and Biophysical Research Communications. 400 (4), 613-618 (2010).</li><li>Boyd, W. A., 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=Developmental+Effect+of+the+ToxCast+Phase+I+and+Phase+II+Chemicals+in+Caenorhabditis+elegans+and+Corresponding+Responses+in+Zebrafish,+Rats,+and+Rabbits.">Developmental Effect of the ToxCast Phase I and Phase II Chemicals in Caenorhabditis elegans and Corresponding Responses in Zebrafish, Rats, and Rabbits.</a> Environmental Health Perspectives. 124 (5), 586-593 (2016).</li></ol>

线虫的优点导致其在毒理学9中的应用越来越多, 无论是在机械研究还是高通量筛选方法。近年来, 线虫在补充其他毒理学研究模型系统方面的作用显著增加, 特别是在新化学品的快速毒性评估方面。本文为384井板蠕虫表型的高通量定量筛选提供了一种新的方法, 用于化学毒性的自动鉴定和评价。该检测方法是24小时内化学品急性毒性检测的理想选择, 可应用于亚急性毒...

随着化合物在工业生产和人们日常生活中的迅速发展, 研究这些化学品的毒性检测模型具有重要意义。在许多情况下, 使用啮齿类动物模型来评估不同化学品对健康的潜在毒性。一般来说, 在体内的啮齿类动物模型中, 确定致死浓度 (即不同化学品的50% 致死剂量 [ld50]) 被用作传统参数, 这既耗时又非常昂贵。此外, 由于减少、提炼或取代 (3r) 原则, 这对动物福利和伦理至关重要, 允许替换更高动物的新方法对科学研究有价值 1、2、3.线虫是一种自由生活的线虫, 已从土壤中分离出来。由于其寿命短、栽培方便、繁殖效率高等优点, 在实验室中得到了广泛的应用。此外, 许多基本的生物途径, 包括基本的生理过程和在线虫的应激反应,在高级哺乳动物4,5,6,7被保存,8. 在我们和其他人进行的几次比较中, 在啮齿类动物中观察到的线虫毒性和毒性之间有很好的一致性9。所有这些都使线虫成为测试体内化学毒性影响的良好模型。
近年来, 一些研究对线虫的表型特征进行了量化。这些特性可用于分析化学品 2、3、10 的毒性和蠕虫的老化11。我们还开发了一种结合液体蠕虫养殖系统和图像分析系统的方法, 在不同的化学处理下, 将蠕虫培养在384孔板中12。该定量技术已开发出来, 可在384孔板中进行12-24h 的化学处理后, 自动分析线虫的33个参数。采用自动显微镜级进行实验视频采集。这些视频由定制设计的程序处理, 与蠕虫移动行为相关的33个功能被量化。该方法用于对10种化合物的处理下的蠕虫表型进行量化。结果表明, 不同的毒性能改变线虫的表型。这些量化的表型可用于识别和预测不同化合物的急性毒性。该方法的总体目标是促进液体培养中线虫实验的观察和表型定量。该方法可用于化学毒性评价和表型定量, 有助于预测不同化合物的急性毒性, 并为进一步的传统化合物建立优先清单。啮齿类动物模型中的化学毒性评估试验。此外, 该方法还可作为食品添加剂污染、药理化合物、环境外源化合物等, 应用于新型化学品或化合物的毒性筛选和检测。

<table border="0" cellpadding="0" cellspacing="0" style="width:959px;" width="957">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:207px;">2-Propanol</td>
			<td style="width:171px;">Sigma-Aldrich</td>
			<td style="width:175px;">59300</td>
			<td style="width:407px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">384-well plates</td>
			<td style="width:171px;">Throme</td>
			<td style="width:175px;">142761</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Agar</td>
			<td>Bacto</td>
			<td style="width:175px;">214010</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:207px;">Atropine sulfate</td>
			<td style="width:171px;">Sigma-Aldrich</td>
			<td style="width:175px;">PHL80892</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:207px;">Bleach buffer</td>
			<td style="width:171px;"></td>
			<td style="width:175px;"></td>
			<td>0.5 mL of 10 M NaOH, 0.5 mL of5% NaClO, 9 mL ofultrapure water</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:207px;">Cadmium chloride</td>
			<td style="width:171px;">Sigma-Aldrich</td>
			<td style="width:175px;">202908</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:207px;">Calcium chloride</td>
			<td style="width:171px;">Sigma-Aldrich</td>
			<td style="width:175px;">21074</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:207px;">CCD camera</td>
			<td style="width:171px;">Zeiss</td>
			<td style="width:175px;">AxioCam HRm</td>
			<td style="width:407px;">Zeiss microscopy GmbH</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:207px;">Cholesterol</td>
			<td style="width:171px;">Sigma-Aldrich</td>
			<td style="width:175px;">C8667</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:207px;">Copper(II) sulfate</td>
			<td style="width:171px;">Sigma-Aldrich</td>
			<td style="width:175px;">451657</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:207px;">Ethanol</td>
			<td style="width:171px;">Sigma-Aldrich</td>
			<td style="width:175px;">24105</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:207px;">Ethylene glycol</td>
			<td style="width:171px;">Sigma-Aldrich</td>
			<td style="width:175px;">324558</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:207px;">Glycerol</td>
			<td style="width:171px;">Sigma-Aldrich</td>
			<td style="width:175px;">G5516</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:207px;">K-Medium</td>
			<td style="width:171px;"></td>
			<td style="width:175px;"></td>
			<td style="width:407px;">3.04 g of NaCl and 2.39 g of KCl in 1 L ultrapure water</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:207px;">LB Broth&#160;</td>
			<td style="width:171px;"></td>
			<td style="width:175px;"></td>
			<td style="width:407px;">10&#160;g/L&#160;Tryptone,&#160;5&#160;g/L&#160;Yeast&#160;Extract,&#160;5&#160;g/L&#160;NaCl&#160;</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:207px;">Magnesium sulfate heptahydrate</td>
			<td style="width:171px;">Sigma-Aldrich</td>
			<td style="width:175px;">63140</td>
			<td></td>
		</tr>
		<tr height="84">
			<td height="84" style="height:84px;width:207px;">NGM Plate</td>
			<td style="width:171px;"></td>
			<td style="width:175px;"></td>
			<td style="width:407px;">3 g ofNaCl, 17 g ofagar, 2.5 g ofpeptone in 1 L of ultrapure water, after autoclave add 1 mL of cholesterol (5 mg/mL in ethanol), 1 mL of MgSO4 (1 M), 1 mL of CaCl2 (1 M), 25 mL of PPB buffer</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Peptone</td>
			<td>Bacto</td>
			<td style="width:175px;">211677</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:207px;">Potassium chloride</td>
			<td style="width:171px;">Sigma-Aldrich</td>
			<td style="width:175px;">60130</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:207px;">Potassium phosphate dibasic</td>
			<td style="width:171px;">Sigma-Aldrich</td>
			<td style="width:175px;">795496</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:207px;">Potassium phosphate monobasic</td>
			<td style="width:171px;">Sigma-Aldrich</td>
			<td style="width:175px;">795488</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:207px;">PPB buffer</td>
			<td style="width:171px;"></td>
			<td style="width:175px;"></td>
			<td style="width:407px;">35.6 g of K2HPO4, 108.3 g of KH2PO4 in 1 L ultrapure water</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:207px;">shaker</td>
			<td style="width:171px;">ZHICHENG</td>
			<td style="width:175px;">ZWY-200D</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:207px;">Sodium chloride</td>
			<td style="width:171px;">Sigma-Aldrich</td>
			<td style="width:175px;">71382</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:207px;">Sodium fluoride</td>
			<td style="width:171px;">Sigma-Aldrich</td>
			<td style="width:175px;">s7920</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:207px;">Sodium hydroxide</td>
			<td style="width:171px;">Sigma-Aldrich</td>
			<td style="width:175px;">71690</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:207px;">Sodium hypochlorite solution</td>
			<td style="width:171px;">Sigma-Aldrich</td>
			<td style="width:175px;">239305</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:207px;">The link of program</td>
			<td style="width:171px;"></td>
			<td style="width:175px;"></td>
			<td style="width:407px;">https://github.com/weiyangc/ImageProcessForWellPlate</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Tryptone</td>
			<td style="width:171px;">Sigma-Aldrich</td>
			<td style="width:175px;">T7293</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Yeast extract</td>
			<td style="width:171px;">Sigma-Aldrich</td>
			<td style="width:175px;">Y1625</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:207px;">Zeiss automatic microscope&#160;</td>
			<td style="width:171px;">Zeiss</td>
			<td style="width:175px;">AXIO Observer.Z1</td>
			<td style="width:407px;">Zeiss automatic microsco with peproprietary software Zen2012 and charge coupled device(CCD) camera</td>
		</tr>
	</tbody>
</table>

a high-throughput assay for the prediction of chemical toxicity by automated phenotypic profiling of caenorhabditis elegans

对小鼠或大鼠等高阶生物中的化学品进行毒性测试是耗时和昂贵的, 因为它们的寿命长, 维护问题。相反, 线虫具有其优势, 使其成为毒性试验的理想选择: 寿命短、易于栽培、繁殖效率高。在这里, 我们描述了一个协议的自动表型分析的线虫在384孔板。线虫在384孔板中进行液体介质和化学处理, 并对每口井进行视频处理, 以量化化学对33种蠕虫特征的影响。实验结果表明, 量化的表型特征可以对不同化合物的急性毒性进行分类和预测, 并为啮齿类动物模型中的进一步传统化学毒性评价试验确定优先清单。

我们已经测试了暴露在超过10种化学物质12的不同浓度下的蠕虫的表型。在测试中, 在三个时间点 (0小时、12小时和 24小时) 对每种化合物的33个不同特征进行了量化。此前, 对人工和寿命分析的自动分析进行了 11、1 2 项比较。在这个试验中, 我们发现化学物质和浓度会影响蠕虫的表型。此方法的概述如<strong class="...

Watch this Scientific Journal Video about A High-throughput Assay for the Prediction of Chemical Toxicity by Automated Phenotypic Profiling of Caenorhabditis elegans at JoVE.com

A High-throughput Assay for the Prediction of Chemical Toxicity by Automated Phenotypic Profiling of Caenorhabditis elegans

通过对线虫表型分析的自动分析, 提出了一种定量鉴别和预测化学品急性毒性的方法。该协议描述了如何在384孔板中使用化学物质处理蠕虫, 捕获视频, 并量化与毒理学相关的表型。

一种用自动表型分析预测线虫化学毒性的高通量测定

Applying toxicity testing of chemicals in higher order organisms, such as mice or rats, is time-consuming and expensive, due to their long lifespan and ...

C.elegans 毒理学分析是有价值的。该协议描述了如何用384井板中的化学物质治疗C.elegans，捕捉视频，并量化毒理学相关的表型。这种方法可以帮助识别和预测一种化学品的潜在急性毒性。这种定量技术已经开发出来，在用液体介质的384井板中经过12至24小时的化学处理后，自动分析C.elegans的33个参数。C.elegans 作为新化学品快速毒性评估的好模型具有价值。作为小鼠，可应用于新化学品的毒性筛选和检测，或将化合物作为食品添加剂污染、药物化合物、环境外源化合物等。仔细阅读协议，并始终遵循一步一步，那么你将是一个很好的。可视化演示便于用户查看每个步骤的操作以及操作结果。演示程序将是张文静和高超韩。他们是我实验室的研究生。首先，对一种新化学品进行初步蠕虫杀伤力测试，以确定最高剂量和最低剂量，即最低浓度为100%致命性，最大浓度为100%非致命性。对于这个实验，准备七个梯度浓度的氯化镉。要准备两次浓度最高的水溶液，在充满8毫升K介质的离心管中溶解92.8毫克氯化镉固体粉末。漩涡混合。粉末完全溶解后，使用移液器填充多达 10 毫升。使用 K 介质稀释，准备其他浓度水平。在超级干净的长凳上，使用无菌尖端从条纹板中挑选一个大肠杆菌OP50的菌落，并放入100毫升LB汤的烧瓶中，为菌落接种。在孵化器摇床中，在37摄氏度下一夜生长。将 NGM 倒入 90 毫升塑料培养板中。每个板的种子与300微升的大肠杆菌OP50溶液。在20摄氏度的NGM板上孵育N2蠕虫约2至3天，直到大多数蠕虫达到成人阶段。用无菌水，将格子蠕虫洗入15毫升无菌锥形离心管中收获。让蠕虫沉淀至少2分钟。吸水，加入五毫升的漂白剂缓冲液。旋转管5分钟，然后将管子放入离心机，以1300的相对离心力旋转30秒，使鸡蛋下粒。使用移液器吸气上流水。将五毫升消毒水加入管中清洗鸡蛋，并旋转管五秒钟。在相对离心力为1300时，将管子离心30秒。取出上一液，加入五毫升消毒水再次清洗。用OP50将鸡蛋移到新的NGM板上。在20摄氏度下孵育过夜。第二天早上，孵化的蠕虫到达L1阶段。大约 40 小时后，蠕虫到达 L4 阶段。使用 K 介质，将 Petri 板的 L4 级蠕虫洗入 50 毫升无菌锥形管中。移液 50 微升的蠕虫液体从管子到玻璃滑梯。在立体显微镜下，使用移液器将蠕虫的浓度调整为每100微升K介质约40只动物。将 50 微升的已准备好的介质添加到 384 井板的每个井中。这些同步的L4级蠕虫已准备好进行以下化学品处理。在添加化学品之前，将 384 井板（与同步蠕虫一起放在自动舞台上）放置，并设置带编程采集程序的摄像机。在384孔板中，蠕虫处理每剂6至7种，8个平行井含有50微升的2倍化学溶液，每个剂量。准备至少三组八组K介质平行井作为控制。为每井添加 50 微升的两倍化学溶液。将时间设置为零点。然后，将384井板放入孵化器摇床中，温度为20摄氏度，每分钟80转。经过所需时间后，从培养箱中取出板，并将其转移到自动阶段。在 12 小时和 24 小时内拍摄每个井的视频，以检查蠕虫的表型。要开始处理实验视频，请将视频文件传输到计算机。通过图形用户界面，单击"选择"按钮，选择源图像目录。添加中间结果目录。中间的结果包括分段图像，这些图像对于处理过的图像的视觉观察非常有用。在界面中添加最终结果目录。在界面中的蠕虫大小文本框中，将平均蠕虫大小参数设置为 2，000。将接口中的移动比率阈值设置为 0.93。单击"分析"按钮开始图像处理。开发的程序可以识别蠕虫并自动量化表型。在本次实验中，对氯化镉处理在三个时间点、零小时、12小时和24小时进行了33个显著特征的量化。实验图像显示，随着化学浓度的增加，蠕虫死亡的速度更快。一开始，控制与化学处理之间没有显著区别。经过12个小时的治疗，高浓度治疗的蠕虫比低浓度或对照组更直，弯曲程度更低。蠕虫体覆盖区域的主要轴长度随着时间的推移而增加。在主轴和小轴长度中，化学浓度均呈从低到高的梯度趋势。根据面积和比例，蠕虫的动性表现出类似的模式。开始时没有观察到显著差异。随着时间的流逝，对照组中的蠕虫显示出活动性的稳定下降。12和24小时后，与对照组相比，用氯化镉治疗的蠕虫在活动性上表现出显著差异。此外，与浓度较低的蠕虫相比，浓度较高的治疗下的蠕虫表现出较弱的活力。总之，该技术为多个领域的快速毒性评估铺平了道路。研究人员可将该方法应用于食品毒性中毒的应急分析、药物化合物的安全评价、急性毒性筛查、新化学品的检测和环境外源化合物。氯化镉是一种低毒物质。处理过程中请戴上手套和口罩，并遵循操作说明。

Research

JoVE Journal

Environment

A High-throughput Assay for the Prediction of Chemical Toxicity by Automated Phenotypic Profiling of Caenorhabditis elegans

Determination of Microbial Extracellular Enzyme Activity in Waters, Soils, and Sediments using High Throughput Microplate Assays

在使用高通量微孔板测定水，土壤，沉积物和微生物测定胞外酶活性的

Much of the nutrient cycling and carbon  processing in natural environments occurs through the activity of extracellular  enzymes released by ...

科研

环境科学

High-throughput Fluorometric Measurement of Potential Soil Extracellular Enzyme Activities

潜在土壤胞外酶活性的高通量荧光测量

Microbes in soils and other environments produce extracellular enzymes to depolymerize and hydrolyze organic macromolecules so that they can be ...

Optimized Negative Staining: a High-throughput Protocol for Examining Small and Asymmetric Protein Structure by Electron Microscopy

优化负染：通过电子显微镜检查小和不对称蛋白结构的高通量协议

Structural determination of proteins is rather challenging for proteins with molecular masses between 40 - 200 kDa. Considering that more than half of ...

Physical, Chemical and Biological Characterization of Six Biochars Produced for the Remediation of Contaminated Sites

物理，化学和六Biochars生产的生物表征污染场地修复的

The physical and chemical properties of biochar vary based on feedstock sources and production conditions, making it possible to engineer biochars with ...

High-throughput Screening of Recalcitrance Variations in Lignocellulosic Biomass: Total Lignin, Lignin Monomers, and Enzymatic Sugar Release

总木质素，木质素单体，和酶促糖推出：顽拗变奏曲在木质纤维素生物质的高通量筛选

The conversion of lignocellulosic biomass to fuels, chemicals, and other commodities has been explored as one possible pathway toward reductions in the ...

High-throughput, Microscale Protocol for the Analysis of Processing Parameters and Nutritional Qualities in Maize (Zea mays L.)

High-throughput, Microscale Protocol for the Analysis of Processing Parameters and Nutritional Qualities in Maize Zea mays L.

玉米加工参数和营养品质分析的高通量、微尺度协议 (玉蜀黍)

Maize is an important grain crop in the United States and worldwide. However, maize grain must be processed prior to human consumption. Furthermore, whole ...

A 3-dimensional (3D)-printed Template for High Throughput Zebrafish Embryo Arraying

A 3-dimensional 3D-printed Template for High Throughput Zebrafish Embryo Arraying

3维 (3 d) 印制的高吞吐量斑马鱼胚胎排列模板

The zebrafish is a globally recognized fresh water organism frequently used in developmental biology, environmental toxicology, and human disease related ...

High-throughput Siderophore Screening from Environmental Samples: Plant Tissues, Bulk Soils, and Rhizosphere Soils

从环境样品中筛选高通量的安德森菌: 植物组织、散装土壤和根际土壤

Siderophores (low-molecular weight metal chelating compounds) are important in various ecological phenomenon ranging from iron (Fe) biogeochemical cycling ...

Using Caenorhabditis elegans for Studying Trans- and Multi-Generational Effects of Toxicants

Using Caenorhabditis elegans for Studying Trans- and Multi-Generational Effects of Toxicants

使用卡内哈布迪炎酶研究有毒物质的跨代和多代效应

Information about toxicities of chemicals are essential in their application and waste management. For chemicals at low concentrations, the long-term ...

Standardized Methods for Measuring Induction of the Heat Shock Response in Caenorhabditis elegans

Standardized Methods for Measuring Induction of the Heat Shock Response in Caenorhabditis elegans

卡诺哈布迪蒂斯精英热休克反应的测量诱导标准化方法

The heat shock response (HSR) is a cellular stress response induced by cytosolic protein misfolding that functions to restore protein folding homeostasis, ...