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 border="0" cellpadding="0" cellspacing="0" width="654">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Safety glasses</td>
			<td style="width:129px;">Uline</td>
			<td style="width:119px;">S-21076&#160;</td>
			<td style="width:191px;"></td>
		</tr>
		<tr height="84">
			<td height="84" style="height:84px;width:216px;">Protective heat resistant glove</td>
			<td style="width:129px;">Grainger</td>
			<td style="width:119px;">Item # 3AT17 Mfr. Model # 3AT17 Catalog Page # 1703</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">50 mL conical tube</td>
			<td style="width:129px;">Falcon</td>
			<td style="width:119px;">14-432-22</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Synthetic Nylon mesh</td>
			<td style="width:129px;">Dynamic 
			Aqua-Supply Ltd</td>
			<td style="width:119px;">NTX20 and NTX50</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Cyanoacrylate glue</td>
			<td style="width:129px;">Scotch Super Glue Liquid</td>
			<td style="width:119px;">SAD114</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Pliers</td>
			<td style="width:129px;">Vampliers</td>
			<td style="width:119px;">VMPVT-001-8</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Dremmel tool with circular file</td>
			<td style="width:129px;">Lowe&#39;s</td>
			<td style="width:119px;">Item # 525945 Model # 100-LG</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">FUDR</td>
			<td style="width:129px;">Sigma</td>
			<td style="width:119px;">F0503</td>
			<td></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">M9 chemicals ( NaCl, Na2HPO4, KH2PO4, MgSO4)&#160;</td>
			<td style="width:129px;">Sigma&#160;</td>
			<td style="width:119px;">S7653, RES20908-A7, 1551139, M7506</td>
			<td></td>
		</tr>
		<tr height="126">
			<td height="126" style="height:126px;width:216px;">NGM plate chemicals (Bactopeptone, Agar, KH2PO4, K2HPO4, CaCl2,Cholesterol, Streptomycin)</td>
			<td style="width:129px;">BD Biosciences (bactopeptone) , Lab express (agar), Sigma ( rest)</td>
			<td style="width:119px;">BD bioscience 211677, Lab Express 1001,&#160; Sigma 1551139, 1551128, C1016, C8667, S6501</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Pluronic F-127</td>
			<td style="width:129px;">Sigma&#160;</td>
			<td style="width:119px;">P2443&#160;</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Paraquat dichloride hydrate</td>
			<td style="width:129px;">Sigma</td>
			<td style="width:119px;">36541&#160;</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Inverted fluorescence microscope</td>
			<td>Olympus&#160;</td>
			<td style="width:119px;">FSX100&#160;</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

Comprehensive Assessment of Germline Chemical Toxicity Using the Nematode Caenorhabditis elegans

Identifying the reproductive toxicity of the thousands of chemicals present in our environment has been one of the most tantalizing challenges in the field of environmental health. This is due in part to the paucity of model systems that can (1) accurately recapitulate keys features of reproductive processes and (2) do so in a medium- to high-throughput fashion, without the need for a high number of vertebrate animals.
We describe here an assay in the nematode C. elegans that allows the rapid identification of germline toxicants by monitoring the induction of aneuploid embryos. By making use of a GFP reporter line, errors in chromosome segregation resulting from germline disruption are easily visualized and quantified by automated fluorescence microscopy. Thus the screening of a particular set of compounds for its toxicity can be performed in a 96- to 384-well plate format in a matter of days. Secondary analysis of positive hits can be performed to determine whether the chromosome abnormalities originated from meiotic disruption or from early embryonic chromosome segregation errors. Altogether, this assay represents a fast first-pass strategy for the rapid assessment of germline dysfunction following chemical exposure.

Comprehensive Assessment of Germline Chemical Toxicity Using the Nematode Caenorhabditis elegans

We describe the detailed steps of a high-throughput chemical assay in the nematode Caenorhabditis elegans used to assess germline toxicity. In this assay, disruption of germline function following chemical exposure is monitored using a fluorescent reporter specific to aneuploid embryos.

生殖系化学毒性的利用线虫综合评估<em&gt;秀丽隐杆线虫</em

Identifying the reproductive toxicity of the thousands of chemicals present in our environment has been one of the most tantalizing challenges in the ...

科研

发育生物学

An Enzymatic Method to Rescue Mesenchymal Stem Cells from Clotted Bone Marrow Samples

Mesenchymal stem cells (MSCs) - usually obtained from bone marrow - often require expansion culture. Our protocol uses clinical grade urokinase to degrade clots in the bone marrow and release MSCs for further use. This protocol provides a rapid and inexpensive alternative to bone marrow resampling. Bone marrow is a major source of MSCs, which are interesting for tissue engineering and autologous stem cell therapies. Upon withdrawal bone marrow may clot, as it comprises all of the hematopoietic system. The resulting clots contain also MSCs that are lost for expansion culture or direct stem cell therapy. We experienced that 74% of canine bone marrow samples contained clots and yielded less than half of the stem cell number expected from unclotted samples. Thus, we developed a protocol for enzymatic digestion of those clots to avoid labor-intense and costly bone marrow resampling. Urokinase - a clinically approved and readily available thrombolytic drug &#8211; clears away the bone marrow clots almost completely. As a consequence, treated bone marrow aspirates yield similar numbers of MSCs as unclotted samples. Also, after urokinase treatment the cells kept their metabolic activity and the ability to differentiate into chondrogenic, osteogenic and adipogenic lineages. Our protocol salvages clotted blood and bone marrow samples without affecting the quality of the cells. This obsoletes resampling, considerably reduces sampling costs and enables the use of clotted samples for research or therapy.

Mesenchymal stem cells are usually obtained from bone marrow and require expansion culture. When samples clot before processing, a protocol using the (enzymatic) thrombolytic drug urokinase can be applied to degrade the clot. Thus, cells are released and available for expansion culture. This protocol provides a rapid and inexpensive alternative to resampling.

酶法从凝块骨髓样本拯救间充质干细胞

Mesenchymal stem cells (MSCs) - usually obtained from bone marrow - often require expansion culture. Our protocol uses clinical grade urokinase to degrade ...

Step-specific Sorting of Mouse Spermatids by Flow Cytometry

The differentiation of mouse spermatids is one critical process for the production of a functional male gamete with an intact genome to be transmitted to the next generation. So far, molecular studies of this morphological transition have been hampered by the lack of a method allowing adequate separation of these important steps of spermatid differentiation for subsequent analyses. Earlier attempts at proper gating of these cells using flow cytometry may have been difficult because of a peculiar increase in DNA fluorescence in spermatids undergoing chromatin remodeling. Based on this observation, we provide details of a simple flow cytometry scheme, allowing reproducible purification of four populations of mouse spermatids fixed with ethanol, each representing a different state in the nuclear remodeling process. Population enrichment is confirmed using step-specific markers and morphological criterions. The purified spermatids can be used for genomic and proteomic analyses.

We describe a sorting strategy for mouse spermatids using flow cytometry. Spermatids are sorted into four highly pure populations, including round (spermiogenesis steps 1-9), early elongating (spermiogenesis steps 10-12), late elongating (spermiogenesis steps 13-14) and elongated spermatids (spermiogenesis steps 15-16). DNA staining, size and granulosity are used as selection parameters.

步特定老鼠的精子细胞分选流式细胞仪

The differentiation of mouse spermatids is one critical process for the production of a functional male gamete with an intact genome to be transmitted to ...

Epicardial Outgrowth Culture Assay and Ex Vivo Assessment of Epicardial-derived Cell Migration

A single layer of epicardial cells lines the heart, providing paracrine factors that stimulate cardiomyocyte proliferation and directly contributing cardiovascular progenitors during development and disease. While a number of factors have been implicated in epicardium-derived cell (EPDC) mobilization, the mechanisms governing their subsequent migration and differentiation are poorly understood. Here, we present in vitro and ex vivo strategies to study EPDC motility and differentiation. First, we describe a method of obtaining primary epicardial cells by outgrowth culture from the embryonic mouse heart. We also introduce a detailed protocol to assess three-dimensional migration of labeled EPDC in an organ culture system. We provide evidence using these techniques that genetic deletion of myocardin-related transcription factors in the epicardium attenuates EPDC migration. This approach serves as a platform to evaluate candidate modifiers of EPDC biology and could be used to develop genetic or chemical screens to identify novel regulators of EPDC mobilization that might be useful for cardiac repair.

Epicardial Outgrowth Culture Assay and Ex Vivo Assessment of Epicardial-derived Cell Migration

Here, we describe methods for isolating primary mouse epicardial cells by an outgrowth culture assay and assessing the functional migration of epicardial-derived cells (EPDC) using an ex vivo heart culture system. These protocols are suitable for identifying genetic and chemical modulators of epicardial epithelial-to-mesenchymal transition (EMT) and motility.

心外膜生长培养测定和<em&gt;离体</em&gt;心外膜源性细胞迁移的评估

A single layer of epicardial cells lines the heart, providing paracrine factors that stimulate cardiomyocyte proliferation and directly contributing ...

Mechanism of Regulation of Adipocyte Numbers in Adult Organisms Through Differentiation and Apoptosis Homeostasis

Considering that adipose tissue (AT) is an endocrine organ, it can influence whole body metabolism. Excessive energy storage leads to the dysregulation of adipocytes, which in turn induces abnormal secretion of adipokines, triggering metabolic syndromes such as obesity, dyslipidemia, hyperglycemia, hyperinsulinemia, insulin resistance and type 2 diabetes. Therefore, investigating the molecular mechanisms behind adipocyte dysregulation could help to develop novel therapeutic strategies. Our protocol describes methods for evaluating the molecular mechanism affected by hypoxic conditions of the AT, which correlates with adipocyte apoptosis in adult mice. This protocol describes how to analyze AT in vivo through gene expression profiling as well as histological analysis of adipocyte differentiation, proliferation and apoptosis during hypoxia exposure, ascertained through staining of hypoxic cells or HIF-1&#945; protein. Furthermore, in vitro analysis of adipocyte differentiation and its responses to various stimuli completes the characterization of the molecular pathways behind possible adipocyte dysfunction leading to metabolic syndromes.

Adipose tissue (AT) can influence whole body homeostasis, therefore understanding the molecular mechanisms of adipocyte differentiation and function is of importance. We provide a protocol for gaining new insights into these processes by analyzing adipocyte homeostasis, differentiation and hypoxia exposure as a model for induced adipocyte apoptosis.

成人脂肪细胞生物体的数字调控机制，通过分化与凋亡的动态平衡

Considering that adipose tissue (AT) is an endocrine organ, it can influence whole body metabolism. Excessive energy storage leads to the dysregulation of ...

Microinjection for Transgenesis and Genome Editing in Threespine Sticklebacks

The threespine stickleback fish has emerged as a powerful system to study the genetic basis of a wide variety of morphological, physiological, and behavioral phenotypes. The remarkably diverse phenotypes that have evolved as marine populations adapt to countless freshwater environments, combined with the ability to cross marine and freshwater forms, provide a rare vertebrate system in which genetics can be used to map genomic regions controlling evolved traits. Excellent genomic resources are now available, facilitating molecular genetic dissection of evolved changes. While mapping experiments generate lists of interesting candidate genes, functional genetic manipulations are required to test the roles of these genes. Gene regulation can be studied with transgenic reporter plasmids and BACs integrated into the genome using the Tol2 transposase system. Functions of specific candidate genes and cis-regulatory elements can be assessed by inducing targeted mutations with TALEN and CRISPR/Cas9 genome editing reagents. All methods require introducing nucleic acids into fertilized one-cell stickleback embryos, a task made challenging by the thick chorion of stickleback embryos and the relatively small and thin blastomere. Here, a detailed protocol for microinjection of nucleic acids into stickleback embryos is described for transgenic and genome editing applications to study gene expression and function, as well as techniques to assess the success of transgenesis and recover stable lines.

Transgenic manipulations and genome editing are critical for functionally testing the roles of genes and cis-regulatory elements. Here a detailed microinjection protocol for the generation of genomic modifications (including Tol2-mediated fluorescent reporter transgene constructs, TALENs, and CRISPRs) is presented for the emergent model fish, the threespine stickleback.

显微注射转基因的基因组，并在编辑Threespine刺鱼

The threespine stickleback fish has emerged as a powerful system to study the genetic basis of a wide variety of morphological, physiological, and ...

Large-Scale Production of Cardiomyocytes from Human Pluripotent Stem Cells Using a Highly Reproducible Small Molecule-Based Differentiation Protocol

Maximizing the benefit of human pluripotent stem cells (hPSCs) for research, disease modeling, pharmaceutical and clinical applications requires robust methods for the large-scale production of functional cell types, including cardiomyocytes. Here we demonstrate that the temporal manipulation of WNT, TGF-&#946;, and SHH signaling pathways leads to highly efficient cardiomyocyte differentiation of single-cell passaged hPSC lines in both static suspension and stirred suspension bioreactor systems. Employing this strategy resulted in ~ 100% beating spheroids, consistently containing &#62; 80% cardiac troponin T-positive cells after 15 days of culture, validated in multiple hPSC lines. We also report on a variation of this protocol for use with cell lines not currently adapted to single-cell passaging, the success of which has been verified in 42 hPSC lines. Cardiomyocytes generated using these protocols express lineage-specific markers and show expected electrophysiological functionalities. Our protocol presents a simple, efficient and robust platform for the large-scale production of human cardiomyocytes.

Here, we present a robust, fast and scalable cardiomyocyte differentiation protocol for human pluripotent stem cells (hPSCs). Cardiomyocytes derived using this large-scale method can provide sufficient cell numbers for their effective use in human cardiovascular disease modeling, high-throughput drug screening, and potentially clinical applications.

从人多能干细胞使用高度可重复的小分子为基础的分化协议心肌细胞规模化生产

Maximizing the benefit of human pluripotent stem cells (hPSCs) for research, disease modeling, pharmaceutical and clinical applications requires robust ...

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 ...

Isotropic Light-Sheet Microscopy and Automated Cell Lineage Analyses to Catalogue Caenorhabditis elegans Embryogenesis with Subcellular Resolution

Caenorhabditis elegans (C. elegans) stands out as the only organism in which the challenge of understanding the cellular origins of an entire nervous system can be observed, with single cell resolution, in vivo. Here, we present an integrated protocol for the examination of neurodevelopment in C. elegans embryos. Our protocol combines imaging, lineaging and neuroanatomical tracing of single cells in developing embryos. We achieve long-term, four-dimensional (4D) imaging of living C. elegans&#160;embryos with nearly isotropic spatial resolution through the use of Dual-view Inverted Selective Plane Illumination Microscopy (diSPIM). Nuclei and neuronal structures in the nematode embryos are imaged and isotropically fused to yield images with resolution of ~330 nm in all three dimensions. These minute-by-minute high-resolution 4D data sets are then analyzed to correlate definitive cell-lineage identities with gene expression and morphological dynamics at single-cell and subcellular levels of detail. Our protocol is structured to enable modular implementation of each of the described steps and enhance studies on embryogenesis, gene expression, or neurodevelopment.

Here, we present a combinatorial approach using high-resolution microscopy, computational tools, and single-cell labeling in living C. elegans embryos to understand single cell dynamics during neurodevelopment.

亚细胞分辨率的各向同性光片显微镜及细胞自动动力学分析

Caenorhabditis elegans (C. elegans) stands out as the only organism in which the challenge of understanding the cellular origins of an entire nervous ...

Measuring Sperm Guidance and Motility within the Caenorhabditis elegans Hermaphrodite Reproductive Tract

Successful fertilization is fundamental to sexual reproduction, yet little is known about the mechanisms that guide sperm to oocytes within the female reproductive tract. While in vitro studies suggest that sperm of internally fertilizing animals can respond to various cues from their surroundings, the inability to visualize their behavior inside the female reproductive tract creates a challenge for understanding sperm migration and mobility in its native environment. Here, we describe a method using C. elegans that overcomes this limitation and takes advantage of their transparent epidermis. C. elegans males stained with a mitochondrial&#160; dye are mated with adult hermaphrodites, which act as modified females, and deposit fluorescently labeled sperm into the hermaphrodite uterus. The migration and motility of the labeled sperm can then be directly tracked using an epi-fluorescence microscope in a live hermaphrodite. In wild-type animals, approximately 90% of the labeled sperm crawl through the uterus and reach the fertilization site, or spermatheca. Images of the uterus can be taken 1 h after mating to assess the distribution of the sperm within the uterus and the percentage of sperm that have reached the spermatheca. Alternatively, time-lapse images can be taken immediately after mating to assess sperm speed, directional velocity and reversal frequency. This method can be combined with other genetic and molecular tools available for the C.elegans to identify novel genetic and molecular mechanisms that are important in regulating sperm guidance and motility within the female reproductive tract.

Measuring Sperm Guidance and Motility within the Caenorhabditis elegans Hermaphrodite Reproductive Tract

Sperm must successfully navigate through the oviduct to fertilize an oocyte. Here, we describe an assay for measuring sperm migration within the C. elegans hermaphrodite uterus. This assay can provide quantitative data on sperm distribution within the uterus after mating, as well as on speed, directional velocity, and reversal frequency.

线虫生殖道内精子引导和运动的测定

Successful fertilization is fundamental to sexual reproduction, yet little is known about the mechanisms that guide sperm to oocytes within the female ...