作者想感谢希瑟科里对研究设计的初步贡献, Swarup 博士米特拉对手稿的批判性审查。我们还要感谢迈克尔. 哈里斯博士在制作这一方法时提出的意见、改进和协助。这些菌株由秀丽遗传学中心提供, 由 NIH 研究基础设施项目办公室 (P40 OD010440) 资助。本出版物所报告的研究得到国家卫生研究院全国医学科学院的支持, UL1GM118991、TL4GM118992、RL5GM118990 和机构发展奖 (构想) 由国家卫生研究院全国医学科学院授予编号5P20GM103395-15。内容完全是作者的责任, 不一定代表国家卫生研究院的官方意见。UA 是 AA/《雇佣条例》的雇主和教育机构, 禁止对任何个人进行非法歧视: www.alaska.edu/titleIXcompliance/nondiscrimination。

1.秀丽筛的施工与使用
<ol><li>
施工协议
	<ol>
<li>从50毫升圆锥管获得2盖子 (图 1A)。</li>
		<li>使用本生燃烧器和热金属探针或烙铁或阶梯钻头, 从底部的内唇中取出中心区域 (当从底端看图 1B)。 注: 使用热切割塑料盖子是最好的刀片, 因为有更少的伤害风险。</li>
		<li>用弯曲的文件或旋转研磨工具 (如Dremel) 清洁和打磨切割的边缘和表面。见图 1C。</li>
		<li>将单丝网格的圆切到适当的直径 (图 1D)。为此, 在一条单丝尼龙网板上跟踪一个盖子, 并在绘制的线内切割。</li>
		<li>当胶水应用于塑料时, 将凹槽/斜线插入盖子的顶面, 以增强两个盖子的附着力 (图 1E)。</li>
		<li>用乙醇清洁盖子, 让它们干。</li>
		<li>将氰胶水涂在两个盖子的顶面, 保持到外边缘。 注意: 小胶水有很长的路要走。</li>
		<li>根据表 1, 将单丝网格放在一个胶合盖子上 (图 1F)。将第二盖子倒在网格的顶部;两个盖子必须有他们的顶端在一起。紧紧地压在一起 (图 1G)。确保网格绷紧。 注: 作为安全措施, 使用镊子将网格放在盖子上。</li>
		<li>一旦胶水的初始层干了, 就在盖子之间的外间隙涂上一圈氰胶。大方, 因为这增加了完整性, 防止任何剥离或泄漏。</li>
		<li>用单丝网孔尺寸将过滤器贴上标签。 注: 在这里, 我们使用两个网孔 sizes—20µm 和50µm。</li>
	</ol>
</li>
	<li>
使用协议
	<ol>
<li>预湿筛。<ol>
<li>吸管一个盐水溶液, 如 M9 [5 克氯化钠, 6 克 Na2HPO4, 3 克的 2 PO 4, 1升的超纯 H2O 和1毫升的 MgSO4 (1 M)]17, 通过筛的中心, 直到液滴形成, 凝结, 并从过滤器底部的中心滴下 (图 2A)。或者, 将不起毛的擦拭 (例如, Kimwipe) 放在底部, 在网格上形成或散布一个湿气滴。</li>
			<li>把筛子放在50毫升圆锥管上。将管标为 "废物管" (图 2B)。</li>
		</ol>
</li>
		<li>从琼脂盘上清洗一个群的线虫。<ol>
<li>用 M9 缓冲器冲洗盘子, 一次将含有蠕虫的介质放在单丝网1毫升的上面 (图 2C)。确保在网格的中心进行操作。把盘子里的虫子都洗干净。 注: 一般情况下, 3 毫升的 M9 为60毫米板是足够的。</li>
			<li>为了尽量减少在吹打中丢失的蠕虫的数量, 请使用玻璃巴斯德吸管将蠕虫从盘子中移出并放到网格中心 (根据需要重复)。</li>
			<li>用 M9 缓冲区从顶部冲洗过滤器。再次, 确保在网格的中心操作, 并冲洗该区域的所有蠕虫。清洗尽可能多的时间, 以确保所有的细菌和较小的蠕虫已经通过网格。</li>
		</ol>
</li>
		<li>从筛子上收获大小匹配的动物。<ol>
<li>将一个新的50毫升圆锥管连接到筛顶部, 并将1.2.2.3 的成虫从台阶上的蠕虫朝向新收集管的内部 (图 2D)。</li>
			<li>卸下第一管 (废管), 快速翻转筛板和新管以防止水滴迁移 (图 2E)。 注意: 如果不育是不需要的, 使用不起毛的擦拭 (例如, Kimwipe) 在翻转之前, 从网格底部的灯芯液。</li>
			<li>用 M9 将网格冲洗到新的上50毫升管中 (图 2F)。再次, 在网格中心运行, 并保持在网格底部的水滴。</li>
			<li>允许蠕虫解决或轻轻地旋转下来 (例如, < 16 x g) 约1分钟 (图 2G)。</li>
			<li>从蠕虫颗粒中吸取缓冲液, 最好是 > 0.5 毫升, 或者在不干扰颗粒的情况下尽可能多地去除液体。</li>
			<li>用玻璃巴斯德吸管将蠕虫吸进 NGM 板上, 让它们干。在一个新的板块上的多个水滴的空间, 使他们干得更快 (图 2H)。</li>
		</ol>
</li>
		<li>清洁筛网。<ol>
<li>用乙醇和反渗透水轻轻、彻底地冲洗筛子。让它干吧。</li>
			<li>将其存储在一个干净的容器中, 以便无限期地使用。</li>
			<li>当网格出现 "凹陷" 时, 丢弃它。</li>
		</ol>
</li>
	</ol>
</li>
</ol>2.秀丽筛分选方法的验证
<ol><li>一般维护
	<ol>
<li>为所有实验, 养殖蠕虫在标准线虫成长媒介17 (1 L NGM 包括2.5 克蛋白胨, 17 克琼脂, 3 g 氯化钠, 975 毫升二蒸馏水, 1 毫升5毫克/毫升胆固醇, 1 毫升 1 M CaCl2, 1 毫升的1米 MgSO4, 25 毫升1米 KHPO4, 0.5 毫升的100毫克/毫升链霉素) 在25摄氏度。</li>
	</ol></li>
	<li>实验治疗管理
	<ol>
<li>比较三治疗组: 摘、fluorodeoxyuridine (FUDR) 和秀丽筛。<ol>
<li>对于 FUDR 治疗组, 添加100毫克/毫升 FUDR 到 NGM 培养基, 最终浓度为100微米, 以防止任何后代的生产, 并转移蠕虫每隔一天的新鲜 NGM 板, 以避免食物枯竭。</li>
			<li>对于拾取组, 请使用白金循环手动选择和传输蠕虫。</li>
			<li>对于秀丽筛治疗组, 请遵循步骤1.2 并将蠕虫吸管移到 NGM 板上。</li>
		</ol>
</li>
	</ol></li>
	<li>秀丽筛分率
	<ol>
<li>为了量化筛检对C. 线虫的有效程度, 在25摄氏度 (即产卵后48小时), 将 N2 动物的年龄同步到1天, 然后通过采摘新鲜的 NGM 盘子 (共计 N = 50 或 N = 100 动物每atment 集团)。</li>
		<li>经过24小时的恢复, 将人口转移到新的 NGM 板与秀丽筛后, 上述协议 (见步骤 1.2) 和计数成功转移动物的数量。</li>
		<li>计算百分比收益率, 因为与转移开始时的起始数相比, 转移的动物数量的比率乘以 100 (%)。</li>
	</ol></li>
	<li>Healthspan 化验 注: 评分 healthspan 参数的运动类, 咽泵率, 以及前和后温和触摸反应在天 2, 4, 6 和8的成年年龄同步 N2 动物维持在25摄氏度。<ol>
<li>动力跟踪<ol>
<li>根据基于类的系统 (类 a、B 和 C) 来分配运动分数, 方法如下. 18. 比较三个实验组在统计分析软件中使用序号逻辑统计模型的效果。 注意: a 类个体自发地以正常的、正弦的模式运动。B 类个体在明显的非正弦运动中移动, 可能需要刺激来鼓励运动。C 类个体移动他们的头和/或尾巴响应刺激, 但不能移动横跨琼脂。</li>
		</ol>
</li>
		<li>咽泵率<ol>
<li>在600X 的最终放大率为1分钟的显微镜下, 将动物末端咽球的研磨运动进行计数。</li>
			<li>用α = 0.05 和 Bonferroni 后测试的方法进行统计分析, 并进行单因子 = 0.05。</li>
		</ol>
</li>
		<li>触摸响应<ol>
<li>记录一个温柔的触摸反应和比较三治疗组。根据 Calixto 所描述的方法进行化验. 19。</li>
			<li>记录前和后接触的反应, 轻轻抚摸睫毛采摘垂直横跨尾部或头部 (5x 每, 交替头和尾巴) 的动物。</li>
			<li>将任何运动的相反方向的中风作为1点的比例为0到5的前和后反应。</li>
			<li>进行统计分析与α = 0.05 和 Bonferroni 后测试与α = 0.05 的单向方差计算。</li>
		</ol>
</li>
	</ol></li>
	<li>繁殖力测定
	<ol>
<li>为了确定使用秀丽筛对生殖的影响, 生长年龄同步 N2 动物到2天成年在25摄氏度。</li>
		<li>60 h 在蛋以后放置, 转移动物到新的 NGM 板材与白金挑选或秀丽筛子并且给他们 4 h 恢复 (干燥被筛的板材为 20–30 min)。</li>
		<li>恢复后, 单独将动物通过睫毛 NGM 板, 给他们24小时产卵, 并删除动物。允许每个板块的子代在正常条件下发育25摄氏度, 另24小时。 注: 睫毛采摘是一个人的睫毛固定在巴斯德吸管的末端与指甲油和乙醇消毒。</li>
		<li>计算可行的 F1 生成个体的数量。使用α = 0.05 的 T 检验进行统计分析。 注意: 存活的后代被认为是成功孵化并通过常规幼虫周期开始发育的卵子。</li>
	</ol></li>
	<li>荧光记者应激反应测定
	<ol>
<li>执行三种常用的荧光报告器检测, 以发现潜在的应力标记: DAF-16::GFP 转移到细胞细胞核的应变 [TJ356-zIs356 (pDAF-16::D af-16-gfp;rol-6)]20;hsp-16.2 表达式 [TJ375-gpIs1 (热休克-16.2p:: GFP)]21;和一个 sod-3 表达 [CF1553-muIs84 ([pAD76] sod-3 p:: GFP + 轧辊-6 [su1006]]22。</li>
		<li>为每种化验, 文化年龄同步动物从三治疗小组在20°c 并且审查他们在3天: 使用消极控制 (每天, 转移一组动物手工用白金采摘), 一个正面控制 (在每日依据, 手动转移一组动物与白金采摘加上一个既定的压力源), 和秀丽筛 (通过筛的动物, 并允许他们恢复30分钟 NGM 就在成像之前)。</li>
		<li>在 DAF-16::GFP 化验中, 热休克的阳性对照动物在37摄氏度30分钟前成像20。为 hsp-16.2 化验, 热休克阳性对照动物90分钟, 20 小时前成像21。对于 sod-3 的检测, 在成像22前将100毫米百草枯的阳性对照动物治疗为4小时。</li>
		<li>立即收获的蠕虫与睫毛采摘和装载他们到一个盖玻片与 1 ul 36% 泊洛沙姆407表面活性剂解决方案, 以固定蠕虫。</li>
		<li>将安装的蠕虫与另一盖玻片三明治。将两个盖玻片安装到标准的玻璃显微显微镜幻灯片上, 并在倒置荧光显微镜 (用于总放大倍数 80X) 和 FITC 过滤器的恒定曝光中使用8X 放大倍数图像。</li>
		<li>为了检测三组之间的任何差异在 DAF-16::GFP 化验, 分类的动物根据 DAF-16::GFP 记者的位置 (核如果记者移位到细胞核, 胞浆如果记者位于细胞质, 和中间如果记者位于原子核和细胞质。</li>
		<li>在统计分析软件中使用序号物流统计模型对结果进行比较。对于 hsp-16.2 和 sod-3 表达式检测, 使用单路方差分析法 = 0.05 和 Tukey 的后临时测试与α = 0.05 比较的总荧光的头部区域。</li>
	</ol></li>
</ol>

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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=Stochastic+and+genetic+factors+influence+tissue-specific+decline+in+ageing+C.+elegans.">Stochastic and genetic factors influence tissue-specific decline in ageing C. elegans.</a> Nature. 419 (6909), 808-814 (2002).</li><li>Calixto, A., Chelur, D., Topalidou, I., Chen, X., Chalfie, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Enhanced+neuronal+RNAi+in+C.+elegans+using+SID-1.">Enhanced neuronal RNAi in C. elegans using SID-1.</a> Nature Methods. 7 (7), 554-559 (2010).</li><li>Henderson, S. T., Johnson, T. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=daf-16+integrates+developmental+and+environmental+inputs+to+mediate+aging+in+the+nematode+Caenorhabditis+elegans.">daf-16 integrates developmental and environmental inputs to mediate aging in the nematode Caenorhabditis elegans.</a> Current Biology. 11 (24), 1975-1980 (2001).</li><li>Rea, S. L., Wu, D., Cypser, J. R., Vaupel, J. W., Johnson, T. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+stress-sensitive+reporter+predicts+longevity+in+isogenic+populations+of+Caenorhabditis+elegans.">A stress-sensitive reporter predicts longevity in isogenic populations of Caenorhabditis elegans.</a> Nature Genetics. 37 (8), 894-898 (2005).</li><li>Libina, N., Berman, J. R., Kenyon, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tissue-specific+activities+of+C.+elegans+DAF-16+in+the+regulation+of+lifespan.">Tissue-specific activities of C. elegans DAF-16 in the regulation of lifespan.</a> Cell. 115 (4), 489-502 (2003).</li><li>Keith, S. A., Amrit, F. R., Ratnappan, R., Ghazi, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+C.+elegans+healthspan+and+stress-resistance+assay+toolkit.">The C. elegans healthspan and stress-resistance assay toolkit.</a> Methods. 68 (3), 476-486 (2014).</li><li>Scerbak, C., Vayndorf, E. M., Hernandez, A., McGill, C., Taylor, B. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanosensory+neuron+aging:+Differential+trajectories+with+lifespan-extending+alaskan+berry+and+fungal+treatments+in+Caenorhabditis+elegans.">Mechanosensory neuron aging: Differential trajectories with lifespan-extending alaskan berry and fungal treatments in Caenorhabditis elegans.</a> Frontiers in Aging Neuroscience. 8, 173 (2016).</li><li>Vayndorf, E. 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=Morphological+remodeling+of+C.+elegans+neurons+during+aging+is+modified+by+compromised+protein+homeostasis.">Morphological remodeling of C. elegans neurons during aging is modified by compromised protein homeostasis.</a> npj Aging and Mechanisms of Disease. 2, 16001 (2016).</li><li>Murakami, S., Johnson, T. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+genetic+pathway+conferring+life+extension+and+resistance+to+UV+stress+in+Caenorhabditis+elegans.">A genetic pathway conferring life extension and resistance to UV stress in Caenorhabditis elegans.</a> Genetics. 143 (3), 1207-1218 (1996).</li><li>Abbas, S., Wink, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Green+Tea+Extract+Induces+the+Resistance+of+Caenorhabditis+elegans+against+Oxidative+Stress.">Green Tea Extract Induces the Resistance of Caenorhabditis elegans against Oxidative Stress.</a> Antioxidants (Basel). 3 (1), 129-143 (2014).</li><li>Yanase, S., Hartman, P. S., Ito, A., Ishii, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Oxidative+stress+pretreatment+increases+the+X-radiation+resistance+of+the+nematode+Caenorhabditis+elegans.">Oxidative stress pretreatment increases the X-radiation resistance of the nematode Caenorhabditis elegans.</a> Mutation Research. 426 (1), 31-39 (1999).</li><li>Chung, K., Crane, M. M., Lu, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Automated+on-chip+rapid+microscopy,+phenotyping+and+sorting+of+C.+elegans.">Automated on-chip rapid microscopy, phenotyping and sorting of C. elegans.</a> Nature Methods. 5 (7), 637-643 (2008).</li></ol>

作者没有什么可透露的。提交人声明, 这项研究是在没有任何商业或金融关系的情况下进行的, 这可能被理解为潜在的利益冲突。

在这里, 我们介绍了设计和使用的方便, 有效的秀丽筛作为一个工具, 以排序和维护C. 线虫。这个工具有几个好处, 手工采摘个别动物, 洗涤人口, 化学处理 (例如, FUDR), 和更昂贵的方法分离动物。首先,秀丽筛有效和快速 (少于20分钟) 排序后代从大混合种群的动物 (表 2)。此外, 该工具的使用对动物的 healthspan 没有可检测的毒性作用 (图 3,<s...

线虫蠕虫,秀丽线虫, 是一个首要的模型有机体。除了他们在实验室里培养的直接和受控性质外, 他们的整个基因组都是1序列, 每个细胞的发育命运都是2。由于这些特点,线虫是一种广泛使用的模型有机体的遗传学研究。然而, 随着这些有益特征的出现, 研究者们面临着一些挑战。由于它们的快速生成时间,线虫种群可以迅速耗尽食物和/或成为混合种群的多个世代和发展阶段出现一次。因此, 在固体线虫生长培养基 (NGM) 上进行的实验要求研究人员在细菌性食物来源耗尽和新幼虫发育之前, 将动物物理地移到新鲜的盘子里。这可能是单调乏味的, 因为需要经常转移动物, 以防止实验种群与后代混合。尽管如此, 一些实验需要大量的动物和延长的时间点 (例如, 在成年时期的 DNA 或 RNA 提取)。这就加剧了精确维持同步种群和转移大量动物的挑战。
目前在 NGM 上养殖线虫的方法是将动物从盘子中采摘或洗涤成盘子;化学处理动物 (例如, 与 DNA 复制抑制剂 fluorodeoxyuridine 或 FUDR);或者使用流式细胞仪对多井板中的动物进行排序。采摘涉及使用一个手工具, 用薄白金线或睫毛, 手动转移个人或多个动物3,4。这种方法是准确的, 但需要的技能和时间, 是一个限制的研究涉及大量的动物。采摘也可能对动物造成身体伤害和压力, 可能会使个体受到不自然和不一致的干扰和强迫。洗涤包括用缓冲液冲洗培养皿, 通过玻璃巴斯德吸管将溶液与动物转移到一个新的培养板上。该方法速度快, 效率高, 但不准确, 因为多代和动物发育阶段大量转移。化学处理, 如 FUDR, 可以溶解在培养基中, 以防止后代的生产通过阻断任何 DNA 复制, 从而, 配子生产和卵子的发展。这种方法虽然有效, 但必须在发育成熟后应用, 以不扰乱正常的发育过程, 这意味着在其管理3之前仍然需要转移动物。这种方法还会影响多细胞信号通路, 对动物的年龄产生明显的影响 (例如, 延长寿命或改变 proteostasis), 这取决于使用5的线虫的应变, 6,7,8,9,10。流式细胞术方法将单个的线虫从一个多井板块自动分类转移到另外11。虽然这种方法是非常有效和有效的, 流式细胞术设备是令人望而却步的昂贵和无法进入的许多研究人员。另一种转移动物的方法是使用温度敏感的突变模型, 如fer-15和fem-1, 当温度调整12时, 它们变得不育。虽然使用变种动物在某些情况下是有用的, 这些特定菌株的增长速度比野生型动物慢, 它们依赖于改变的基因组, 作为衰老或健康蠕虫的不良代表。此外, 依赖温度转移诱导不孕也导致没有一个静态环境, 和温度变化已经很容易地显示影响基因表达13,14, 15. 研究小组以前发表过一些技术, 描述使用网格筛选C. 线虫的大小为16。但是, 我们无法找到以前的工作测试, 以了解与使用此类过滤器有关的总体健康结果的任何变化。
因此, 在C. 线虫研究社区中需要一种负担得起的、高效的、快速的、准确的方法, 在培养皿之间转移大量的动物。我们开发了一种改进的、可访问的设备 (命名为秀丽筛), 并为其制造和操作提供了相关的协议, 以满足C. 线虫研究社区的需要。在此, 我们分享秀丽筛的设计和使用方法, 我们证明, 它的使用不影响共同的健康或任何压力标记相比, 标准手工采摘和治疗与常用的,限制生育的化学 FUDR。

<table><tbody><tr><td>Safety glasses</td><td>Uline</td><td>S-21076</td><td></td></tr><tr><td>Protective heat resistant glove</td><td>Grainger</td><td>Item # 3AT17 Mfr. Model # 3AT17 Catalog Page # 1703</td><td></td></tr><tr><td>50 mL conical tube</td><td>Falcon</td><td>14-432-22</td><td></td></tr><tr><td>Synthetic Nylon mesh</td><td>Dynamic&lt;br/&gt; Aqua-Supply Ltd</td><td>NTX20 and NTX50</td><td></td></tr><tr><td>Cyanoacrylate glue</td><td>Scotch Super Glue Liquid</td><td>SAD114</td><td></td></tr><tr><td>Pliers</td><td>Vampliers</td><td>VMPVT-001-8</td><td></td></tr><tr><td>Dremmel tool with circular file</td><td>Lowe&#039;s</td><td>Item # 525945 Model # 100-LG</td><td></td></tr><tr><td>FUDR</td><td>Sigma</td><td>F0503</td><td></td></tr><tr><td>M9 chemicals ( NaCl, Na2HPO4, KH2PO4, MgSO4)&amp;nbsp;</td><td>Sigma&amp;nbsp;</td><td>S7653, RES20908-A7, 1551139, M7506</td><td></td></tr><tr><td>NGM plate chemicals (Bactopeptone, Agar, KH2PO4, K2HPO4, CaCl2,Cholesterol, Streptomycin)</td><td>BD Biosciences (bactopeptone) , Lab express (agar), Sigma ( rest)</td><td>BD bioscience 211677, Lab Express 1001,&amp;nbsp; Sigma 1551139, 1551128, C1016, C8667, S6501</td><td></td></tr><tr><td>Pluronic F-127</td><td>Sigma</td><td>P2443</td><td></td></tr><tr><td>Paraquat dichloride hydrate</td><td>Sigma</td><td>36541</td><td></td></tr><tr><td>Inverted fluorescence microscope</td><td>Olympus</td><td>FSX100</td><td></td></tr></tbody></table>

caenorhabditis sieve: a low-tech instrument and methodology for sorting small multicellular organisms

秀丽线虫(C. 线虫) 是一种在一系列基础和生物医学研究中使用的成熟的模型有机体。在线虫研究社区内, 需要一种负担得起和有效的方法来维持大型、年龄匹配的线虫种群。在这里, 我们提出了机械分拣和清洁线虫的方法。我们的目标是提供一个经济高效, 高效, 快速, 简单的过程, 以获得相同大小和生命阶段的动物在实验中使用。这个工具,秀丽筛, 使用一个定制的盖子系统, 线程到普通锥形实验室管和排序C. 线虫的基础上的体型。我们还证明,秀丽筛有效地转移动物从一个文化板块到另一个允许快速排序, 同步和清洁, 而不影响健康标志, 包括运动和应力诱导基因记者。这种易于使用和创新的工具是一种快速、高效和无压力的选项, 用于维护C. 线虫种群。

秀丽筛由2个螺钉盖组成, 保证了编织尼龙丝网的面积小于所需发育年龄的体径, 用于用简单的洗涤技术提取生物体的活种群。它附着在标准锥形管上, 使用网格屏幕按身体直径对动物进行机械分类, 使所需的动物在试管中准备好进行进一步的维护和试验 (例如, 转移或遗传收获)。一个温和的手工洗涤与秀丽筛是快速的, 约5分钟每60–100毫米板, 和有机体很?...

Watch this Scientific Journal Video about Caenorhabditis Sieve: A Low-tech Instrument and Methodology for Sorting Small Multicellular Organisms at JoVE.com

Caenorhabditis Sieve: A Low-tech Instrument and Methodology for Sorting Small Multicellular Organisms

目前的议定书包括了一个分类和清洁秀丽线虫年龄匹配种群的方法。它使用一个简单, 廉价, 高效的定制工具, 以获得大量的实验种群的线虫进行研究。

秀丽筛网: 一种低技术的小型多细胞生物分类方法

Caenorhabditis elegans (C. elegans) is a well-established model organism used across a range of basic and biomedical research. Within the nematode ...

caenorhabditis-sieve-low-tech-instrument-methodology-for-sorting

Research

JoVE Journal

Developmental Biology

Caenorhabditis Sieve: A Low-tech Instrument and Methodology for Sorting Small Multicellular Organisms

6.2K 次观看.  University of Alaska Fairbanks. 目前的议定书包括了一个分类和清洁秀丽线虫年龄匹配种群的方法。它使用一个简单, 廉价, 高效的定制工具, 以获得大量的实验种群的线虫进行研究。

视频: Caenorhabditis Sieve: A Low-tech Instrument and Methodology for Sorting Small Multicellular Organisms

Culture and Assay of Large-Scale Mixed-Stage Caenorhabditis elegans Populations

Caenorhabditis elegans (C. elegans) has been and remains a valuable model organism to study developmental biology, aging, neurobiology, and genetics. The large body of work on C. elegans makes it an ideal candidate to integrate into large-population, whole-animal studies to dissect the complex biological components and their relationships with another organism. In order to use C. elegans in collaborative -omics research, a method is needed to generate large populations of animals where a single sample can be split and assayed across diverse platforms for comparative analyses.
Here, a method to culture and collect an abundant mixed-stage C. elegans population on a large-scale culture plate (LSCP) and subsequent phenotypic data is presented. This pipeline yields sufficient numbers of animals to collect phenotypic and population data, along with any data needed for -omics experiments (i.e., genomics, transcriptomics, proteomics, and metabolomics). In addition, the LSCP method requires minimal manipulation to the animals themselves, less user preparation time, provides tight environmental control, and ensures that handling of each sample is consistent throughout the study for overall reproducibility. Lastly, methods to document population size and population distribution of C. elegans life stages in a given LSCP are presented.

Culture and Assay of Large-Scale Mixed-Stage Caenorhabditis elegans Populations

To use Caenorhabditis elegans (C. elegans) in omics research, a method is needed to generate large populations of worms where a single sample can be measured across platforms for comparative analyses. Here, a method to culture C. elegans populations on large-scale culture plates (LSCPs) and to document population growth is presented.

大规模混合阶段 卡诺哈布迪蒂斯电子 人种群的文化和分析

Caenorhabditis elegans (C. elegans) has been and remains a valuable model organism to study developmental biology, aging, neurobiology, and genetics. The ...

科研

遗传学

Automated Separation of C. elegans Variably Colonized by a Bacterial Pathogen

The wormsorter is an instrument analogous to a FACS machine that is used in studies of Caenorhabditis elegans, typically to sort worms based on expression of a fluorescent reporter. Here, we highlight an alternative usage of this instrument, for sorting worms according to their degree of colonization by a GFP-expressing pathogen. This new usage allowed us to address the relationship between colonization of the worm intestine and induction of immune responses. While C. elegans immune responses to different pathogens have been documented, it is still unknown what initiates them. The two main possibilities (which are not mutually exclusive) are recognition of pathogen-associated molecular patterns, and detection of damage caused by infection. To differentiate between the two possibilities, exposure to the pathogen must be dissociated from the damage it causes. The wormsorter enabled separation of worms that were extensively-colonized by the Gram-negative pathogen Pseudomonas aeruginosa, with the damage likely caused by pathogen load, from worms that were similarly exposed, but not, or marginally, colonized. These distinct populations were used to assess the relationship between pathogen load and the induction of transcriptional immune responses. The results suggest that the two are dissociated, supporting the possibility of pathogen recognition.

Automated Separation of C. elegans Variably Colonized by a Bacterial Pathogen

The wormsorter facilitates genetic screens in Caenorhabditis elegans by sorting worms according to expression of fluorescent reporters. Here, we describe a new usage:&#160;sorting according to colonization by a GFP-expressing pathogen, and we employ it to examine the poorly understood role of pathogen recognition in initiating immune responses.

自动分离<em&gt; C。线虫</em&gt;可多段由细菌病原体殖民者

The wormsorter is an instrument analogous to a FACS machine that is used in studies of Caenorhabditis elegans, typically to sort worms based on expression ...

免疫与感染

Novel Metrics to Characterize Embryonic Elongation of the Nematode Caenorhabditis elegans

Dissecting the signaling pathways that control the alteration of morphogenic processes during embryonic development requires robust and sensitive metrics. Embryonic elongation of the nematode Caenorhabditis elegans is a late developmental stage consisting of the elongation of the embryo along its longitudinal axis. This developmental stage is controlled by intercellular communication between hypodermal cells and underlying body-wall muscles. These signaling mechanisms control the morphology of hypodermal cells by remodeling the cytoskeleton and the cell-cell junctions. Measurement of embryonic lethality and developmental arrest at larval stages as well as alteration of cytoskeleton and cell-cell adhesion structures in hypodermal and muscle cells are classical phenotypes that have been used for more than 25 years to dissect these signaling pathways. Recent studies required the development of novel metrics specifically targeting either early or late elongation and characterizing morphogenic defects along the antero-posterior axis of the embryo. Here, we provide detailed protocols enabling the accurate measurement of the length and the width of the elongating embryos as well as the length of synchronized larvae. These methods constitute useful tools to identify genes controlling elongation, to assess whether these genes control both early and late phases of this stage and are required evenly along the antero-posterior axis of the embryo.

Novel Metrics to Characterize Embryonic Elongation of the Nematode Caenorhabditis elegans

Here we detail protocols specifically designed to monitor morphogenic defects that occur during early and late phases of embryonic elongation of the nematode Caenorhabditis elegans. Ultimately, these protocols are designed to identify genes that regulate these phases and to characterize their differential requirements along the antero-posterior axis of the embryo.

新颖的指标来表征线虫胚胎伸长率<em&gt;秀丽隐杆线虫</em

Dissecting the signaling pathways that control the alteration of morphogenic processes during embryonic development requires robust and sensitive metrics. ...

神经科学

Basic Caenorhabditis elegans Methods: Synchronization and Observation

Research into the molecular and developmental biology of the nematode Caenorhabditis elegans was begun in the early seventies by Sydney Brenner and it has since been used extensively as a model organism 1. C. elegans possesses key attributes such as simplicity, transparency and short life cycle that have made it a suitable experimental system for fundamental biological studies for many years 2. Discoveries in this nematode have broad implications because many cellular and molecular processes that control animal development are evolutionary conserved 3.
C. elegans life cycle goes through an embryonic stage and four larval stages before animals reach adulthood. Development can take 2 to 4 days depending on the temperature. In each of the stages several characteristic traits can be observed. The knowledge of its complete cell lineage 4,5 together with the deep annotation of its genome turn this nematode into a great model in fields as diverse as the neurobiology 6, aging 7,8, stem cell biology 9 and germ line biology 10. 
An additional feature that makes C. elegans an attractive model to work with is the possibility of obtaining populations of worms synchronized at a specific stage through a relatively easy protocol. The ease of maintaining and propagating this nematode added to the possibility of synchronization provide a powerful tool to obtain large amounts of worms, which can be used for a wide variety of small or high-throughput experiments such as RNAi screens, microarrays, massive sequencing, immunoblot or in situ hybridization, among others. 
Because of its transparency, C. elegans structures can be distinguished under the microscope using Differential Interference Contrast microscopy, also known as Nomarski microscopy. The use of a fluorescent DNA binder, DAPI (4',6-diamidino-2-phenylindole), for instance, can lead to the specific identification and localization of individual cells, as well as subcellular structures/defects associated to them.

Basic Caenorhabditis elegans Methods: Synchronization and Observation

The easiness of maintaining and propagating the nematode C. elegans make it a nice model organism to work with. The possibility of synchronizing worms allows the work with a significant amount of subjects at the same developmental stage, what facilitates the study of one particular process in many animals.

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 ...

生物学

Sorting of Streptomyces Cell Pellets Using a Complex Object Parametric Analyzer and Sorter

Streptomycetes are filamentous soil bacteria that are used in industry for the production of enzymes and antibiotics. When grown in bioreactors, these organisms form networks of interconnected hyphae, known as pellets, which are heterogeneous in size. Here we describe a method to analyze and sort mycelial pellets using a Complex Object Parametric Analyzer and Sorter (COPAS). Detailed instructions are given for the use of the instrument and the basic statistical analysis of the data. We furthermore describe how pellets can be sorted according to user-defined settings, which enables downstream processing such as the analysis of the RNA or protein content. Using this methodology the mechanism underlying heterogeneous growth can be tackled. This will be instrumental for improving streptomycetes as a cell factory, considering the fact that productivity correlates with pellet size.

Sorting of Streptomyces Cell Pellets Using a Complex Object Parametric Analyzer and Sorter

Liquid-grown Streptomyces cultures are characterized by mycelial pellets that are heterogeneous in size. We here describe a method to analyze and sort such pellets in a high-throughput manner. These pellets can be used for further analyses, which will provide leads to understand and control growth heterogeneity.

排序<em&gt;链霉菌</em&gt;细胞沉淀用一个复杂的对象参数分析仪和分选机

Streptomycetes are filamentous soil bacteria that are used in industry for the production of enzymes and antibiotics. When grown in bioreactors, these ...

Automating Aggregate Quantification in Caenorhabditis elegans

A rise in the prevalence of neurodegenerative protein conformational diseases (PCDs) has fostered a great interest in this subject over the years. This increased attention has called for the diversification and improvement of animal models capable of reproducing disease phenotypes observed in humans with PCDs. Though murine models have proven invaluable, they are expensive and are associated with laborious, low-throughput methods. Use of the Caenorhabditis elegans nematode model to study PCDs has been justified by its relative ease of maintenance, low cost, and rapid generation time, which allow for high-throughput applications. Additionally, high conservation between the C. elegans and human genomes makes this model an invaluable discovery tool. Nematodes that express fluorescently tagged tissue-specific polyglutamine (polyQ) tracts exhibit age- and polyQ length-dependent aggregation characterized by fluorescent foci. Such reporters are often employed as proxies to monitor changes in proteostasis across tissues. Manual aggregate quantification is time-consuming, limiting experimental throughput. Furthermore, manual foci quantification can introduce bias, as aggregate identification can be highly subjective. Herein, a protocol consisting of worm culturing, image acquisition, and data processing was standardized to support high-throughput aggregate quantification using C. elegans that express intestine-specific polyQ. By implementing a C. elegans-based image processing pipeline using CellProfiler, an image analysis software, this method has been optimized to separate and identify individual worms and enumerate their respective aggregates. Though the concept of automation is not entirely unique, the need to standardize such procedures for reproducibility, elimination of bias from manual counting, and increase throughput is high. It is anticipated that these methods can drastically simplify the screening process of large bacterial, genomic, or drug libraries using the C. elegans model.

Automating Aggregate Quantification in Caenorhabditis elegans

The following protocol describes the development and optimization of a high-throughput workflow for worm culturing, fluorescence imaging, and automated image processing to quantify polyglutamine aggregates as an assessment of changes in proteostasis.

自动化秀丽隐杆线虫的聚集定量

A rise in the prevalence of neurodegenerative protein conformational diseases (PCDs) has fostered a great interest in this subject over the years. This ...

A Highly Scalable Approach to Perform Ecological Surveys of Selfing Caenorhabditis Nematodes

Caenorhabditis elegans is one of the major model organisms in biology, but only recently have researchers focused on its natural ecology. The relative sparsity of information about C. elegans in its natural context comes from the challenges involved in the identification of the small nematode in nature. Despite these challenges, an increasing focus on the ecology of C. elegans has caused a wealth of new information regarding its life outside of the laboratory. The intensified search for C. elegans in nature has contributed to the discovery of many new Caenorhabditis species and revealed that congeneric nematodes frequently cohabitate in the wild, where they feed on microbial blooms associated with rotting plant material. The identification of new species has also revealed that the androdioecious mating system of males and self-fertilizing hermaphrodites has evolved three times independently within Caenorhabditis. The other two selfing species, C. briggsae and C. tropicalis, share the experimental advantages of C. elegans and have enabled comparative studies into the mechanistic basis of important traits, including self-fertilization. Despite these advances, much remains to be learned about the ecology and natural diversity of these important species. For example, we still lack functional information for many of their genes, which might only be attained through an understanding of their natural ecology. To facilitate ecological research of selfing Caenorhabditis nematodes, we developed a highly scalable method to collect nematodes from the wild. Our method makes use of mobile data collection platforms, cloud-based databases, and the R software environment to enhance researchers&#39; ability to collect nematodes from the wild, record associated ecological data, and identify wild nematodes using molecular barcodes.

A Highly Scalable Approach to Perform Ecological Surveys of Selfing Caenorhabditis Nematodes

This protocol can be used to perform large-scale ecological surveys of selfing Caenorhabditis nematodes. The primary advantage of this method is the efficient organization and analysis of ecological and molecular data associated with the nematodes collected from nature.

一种高度可扩展的方法，用于对自发 性Caenorhabditis 线虫进行生态调查

Caenorhabditis elegans is one of the major model organisms in biology, but only recently have researchers focused on its natural ecology. The relative ...

Laser Microdissection for Species-Agnostic Single-Tissue Applications

Single-cell methodologies have revolutionized the analysis of the transcriptomes of specific cell types. However, they often require species-specific genetic &#34;toolkits,&#34; such as promoters driving tissue-specific expression of fluorescent proteins. Further, protocols that disrupt tissues to isolate individual cells remove cells from their native environment (e.g., signaling from neighbors) and may result in stress responses or other differences from native gene expression states. In the present protocol, laser microdissection (LMD) is optimized to isolate individual nematode tail tips for the study of gene expression during male tail tip morphogenesis.
LMD allows the isolation of a portion of the animal without the need for cellular disruption or species-specific toolkits and is thus applicable to any species. Subsequently, single-cell RNA-seq library preparation protocols such as CEL-Seq2 can be applied to LMD-isolated single tissues and analyzed using standard pipelines, given that a well-annotated genome or transcriptome is available for the species. Such data can be used to establish how conserved or different the transcriptomes are that underlie the development of that tissue in different species.
Limitations include the ability to cut out the tissue of interest and the sample size. A power analysis shows that as few as 70 tail tips per condition are required for 80% power. Tight synchronization of development is needed to obtain this number of animals at the same developmental stage. Thus, a method to synchronize animals at 1 h intervals is also described.

A protocol is described that uses laser microdissection to isolate individual nematode tissues for RNA-sequencing. The protocol does not require species-specific genetic toolkits, allowing gene expression profiles to be compared between different species at the level of single-tissue samples.

激光显微切割，用于与物种无关的单组织应用

Single-cell methodologies have revolutionized the analysis of the transcriptomes of specific cell types. However, they often require species-specific ...

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, ...

Single Cell Dissociation of Caenorhabditis elegans: A Method to Isolate Live Cells

Source: Germany, E. M., et al. <a href="https://www.jove.com/video/60145/isolation-specific-neuron-populations-from-roundworm-caenorhabditis">Isolation of Specific Neuron Populations from Roundworm Caenorhabditis elegans</a>. J. Vis. Exp. (2019).
This video describes a method to dissociate whole worms into a single-cell suspension suitable for FACS or immunoprecipitation of intact cells.

秀丽隐杆线虫的单细胞解离:一种分离活细胞的方法

Source: Germany, E. M., et al. Isolation of Specific Neuron Populations from Roundworm Caenorhabditis elegans. J. Vis. Exp. (2019).
This video describes a ...

秀丽筛网: 一种低技术的小型多细胞生物分类方法

摘要

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此视频中的章节

大规模混合阶段卡诺哈布迪蒂斯电子人种群的文化和分析

自动分离

新颖的指标来表征线虫胚胎伸长率

基本的

排序

自动化秀丽隐杆线虫的聚集定量

一种高度可扩展的方法，用于对自发性Caenorhabditis 线虫进行生态调查

激光显微切割，用于与物种无关的单组织应用

秀丽隐杆线虫肠的手部解剖

秀丽隐杆线虫的单细胞解离:一种分离活细胞的方法

秀丽筛网: 一种低技术的小型多细胞生物分类方法

摘要

探索更多视频

此视频中的章节

大规模混合阶段 卡诺哈布迪蒂斯电子 人种群的文化和分析

自动分离

新颖的指标来表征线虫胚胎伸长率

基本的

排序

自动化秀丽隐杆线虫的聚集定量

一种高度可扩展的方法，用于对自发 性Caenorhabditis 线虫进行生态调查

激光显微切割，用于与物种无关的单组织应用

秀丽隐杆线虫肠的手部解剖

秀丽隐杆线虫的单细胞解离:一种分离活细胞的方法

大规模混合阶段卡诺哈布迪蒂斯电子人种群的文化和分析

一种高度可扩展的方法，用于对自发性Caenorhabditis 线虫进行生态调查