Loredana Manfra环境的保护和研究研究所（伊斯普拉部环境质量监测）区域环境保护署（艾米利亚 - 罗马涅，意大利）和Federica Savorelli产生。 海水/苦咸水和水质的UNICHIM委员会（在化工行业的技术标准化工作的机构）的沉积物- 卤虫甲壳类生物方法集团内的子工作组议员参考。 录像和编辑由马尔科，伊斯普拉Pisapia（网络单位）。 由安娜·玛丽亚·西塞罗和Erika伊斯普拉Magaletti的的环境质量监测部生产协调。 我们要感谢集团的生物方法费拉拉科，艾米利亚 - 罗马涅，环境保护的区域代理 - 海水/苦咸水水和泥沙的UNICHIM委员会对水质和卢西亚娜米廖雷，Tor Vergata大学（罗马）的合作;洛舍Boscolo和马西莫加贝里尼，伊斯普拉（预防和减轻影响）提供财政支持的法比奥Matassa和伦敦在罗马的语言学校，他们的语言支持。

1。方法标准我们的测试，包括卤虫幼虫暴露物质浓度的时间间隔或确定的浓度或稀释会导致50％的生物体死亡（LC 50）超过14天暴露水平的目的为测试样品，并符合定义的条件用这种方法。如果必要和可能，也可确定如下:一）的浓度水平，导致死亡的20％（LC 20）暴露生物体;二）分析的最高浓度水平并不确定死亡率高于阴性对照组（NOEC）;测试的最低浓度水平的决定后14天，比阴性对照组（LOEC）死亡率较高。 2。材料试验 2.1 卤虫卵执行测试， 卤虫franciscana </em&gt;不少于48小时的幼虫（幼体）的要求，在第二或第三阶段，从刚孵出的鸡蛋5获得。用来支持测试认证的鸡蛋或囊肿市售例如，从质量保证研究部，美国环境保护局，辛辛那提OH 45268，USA或污染水产品中，比利时根特大学生物研究实验室。 2.2水稀释作为稀释水，可用于合成海水准备解散公认的分析质量试剂或蒸馏水或去离子水市售的配方。市售创造了理想的海水盐混合物，例如即时海洋混合物。建议用稀释水的盐度等于35（±2）电源供应器，以及超过80％的饱和值的溶解氧浓度和温度为25（±2）度; C.稀释后曝气48小时，并在孔隙率至少0.45微米的过滤器过滤，可存储最多30天在黑暗中，在温度0-4°C。 2.3藻类文化建议测试生物体是杜氏tertiolecta微藻，饲料成倍增长，达到密度1.3x10 6 - 2.0x10 6细胞/ ml。环境样品的测试需要培养密度高于2.0x10 6细胞/ ml 6。建议， 杜氏tertiolecta生长在文化媒体含有海水（30（±2）PSU和20（±1）°），除了必需的营养素，矿物质和维生素。一些迹象表明藻媒体组成和准备报告附录中。建议初始细胞密度约10 5细胞/ ml。在保持藻类文化房间恒温在20（±1）°C间，与荧光灯3000（±300）LX与16小时的光照8小时黑暗的光照比，照亮。 其他浮游植物也可以被用来喂养，但有必要使这些浮游植物的比较研究，以找到"等同于"物种杜氏tertiolecta，因为实验条件（如初始细胞密度，光照，温度，组成浮游植物物种之间的文化传媒和曝光时间）不等。除了 ​​需要考虑不同污染物的耐受性微藻，因为污染物对浮游植物的影响，也对卤虫生长和发展的影响。 2.4对照品在本次测试的参考物质是十二烷基硫酸钠。 2.5实验室玻璃器皿<ol><li>测试容器，包括100毫升的硼硅玻璃烧杯（18进行测试和传输解决方案18）。 </li><li>一瓶酒（如500毫升瓶）准备标准的解决方案。 </li><li>六瓶（如200毫升瓶）编写的测试解决方案。 </li><li>包括被激活囊肿和卤虫孵化盘转移到测试容器使用玻璃或带有可拆卸的聚苯乙烯直径5厘米的培养皿。 </li><li>玻璃巴斯德移液器（火焰四舍五入提示）为转移的幼体。 </li><li>玻璃套管或巴斯德3毫升的一次性塑料移液器为转移卤虫 （削减）。 </li><li>微电极和毕业移液器准备的测试解决方案。 </li><li> 6个量筒（例如，50毫升的气瓶）的转移烧瓶测试解决方案，到T他测试容器。 </li></ol> 3。囊肿激活 3.1。培养皿中含有囊肿+倒水要生成测试生物体，20毫克囊肿放入一个Petri测试前48小时内含有12毫升稀释水菜。 3.2。光在黑暗中孵化培养皿维持在25（±2）℃和（±1000）4000勒克司（流明每平方米）一小时的光照强度。 3.3。更换介质使用显微镜 24小时后，孵化的幼虫被转移到一个新的Petri菜充满了人工水。使用显微镜，即使用一个光源，这样的孵化，的向光性幼虫迁移对光束进行转移。巴斯德玻璃吸管是用来做转移，确保只有新生幼虫ARé转移，而不是囊肿或幼虫仍然在细胞膜。 3.4。孵化中的黑暗含有幼虫的菜被放置在一个黑暗的恒温室（24小时25±2）°C。 4。制备测试解决方案 4.1。称量物质+转移到容量瓶它被推荐为测试物质的标准解决方案，是由溶解在500毫升容量瓶物质0.5Ğ准备。烧瓶充满了去离子水或蒸馏水，搅拌直到完全溶解的测试物质的溶液。解决方案已经在使用时要准备的，除非它不知道是否在溶液中的物质是稳定的。在这种情况下，标准的解决方案可能准备测试前两天。 4.2。准备阴性对照，加入稀释水和藻类的无线网络第10毫升吸管准备在烧瓶（如200毫升体积）的取藻悬浮液中加入稀释水，使10 5细胞/ ml密度得到阴性对照。 4.3。准备测试解决方案:添加稀释水，藻类和标准溶液据建议，准备加入标准的解决方案，所以，检测所需的浓度，得到指定数量的稀释水500 200毫升瓶测试解决方案。养活生物体，的杜氏tertiolecta的微藻暂停等分可能被添加到测试解决方案的准备时，达到10 5细胞/ ml密度。除下列顺序建议:稀释水，藻悬浮液和标准溶液。编写测试解决方案后，他们必须尽快在实验中使用。 4.4。传输瓶测试容器的解决方案，然后向培养皿同等数量的测试解决方案（50毫升）引入测试利用量筒容器。每个浓度的三个副本。对于每个一系列的测试，控制容器的准备与测试解决方案的体积等于稀释水的用量。等量的测试解决方案（约12毫升）被引入到培养皿。对于每个一系列的测试，控制培养皿中被使用。 5。测试的准备工作 5.1。幼体在II-III期囊肿激活后四十八小时，无节幼体达到Ⅱ - Ⅲ幼虫阶段，然后测试可用。 5.2。幼体转移到的Petri菜用显微镜囊肿使用的培养皿<html>激活取出恒温室。少量的幼虫转移到Petri网控制和测试解决方案的菜肴。这个任务是要进行显微镜利用一个足够宽直径的吸管，使生物体不损坏。在这个阶段的测试，可转让巴斯德玻璃吸管（圆尖火焰）和横向定位的光源，鼓励卤虫聚集。 5.3。幼体转移到测试容器的培养皿，在显微镜十幼虫转移到测试容器测试解决方案，从培养皿。此操作也可以利用巴斯德吸管和显微镜进行。据建议，体积不超过1毫升的幼虫通过在不影响测试系统的整体体积。 e_step"&gt; 5.4。容器用石蜡膜覆盖测试烧杯覆盖石蜡膜（留下机票的差距），并保持温度在25（±2）℃，整个测试时间为900（±100）lx的照度与光照14小时的光照至10小时的黑暗中的比例。 6。更换介质及辅食 6.1。成活率控制两天后，已开始测试，然后5，7，9，12天之后， 卤虫验证的成活率和更换介质和辅食的显微镜下观察。 活生物体的数量，在每个测试容器。后在显微镜下观察和轻微的机械刺激（如触摸玻璃巴斯德吸管幼虫）不显示约10秒的一些运动的生物体econds应考虑死亡。 6.2。制备的藻类采样与10毫升吸管和微量测试物质的抽样测试显示解决方案。从烧瓶试验容器传输解决方案测试时，测试解决方案必须定期更换，并在同一天，因为他们要使用准备。测试解决方案是由以前编制的标准溶液。 6.3 卤虫转移，从旧的测试容器，利用切巴斯德吸管，三个新的容器用一个足够大直径的吸管，以免破坏生物体的转让卤虫进行。在此阶段，可用于3毫升塑料巴斯德吸管（削减）。 6.4。在烧杯中的幼虫含有任何活的或死的卤虫 经过1<html>在测试结束4天的接触，存活幼虫的数量计算，并在工作表上的记录。 LC 50和/或LC 20，在14天的NOEC和LOEC，计算和记录，以及可信限在95％的地方，适当和计算方法。 7。代表结果在测试结束（14天），计算每个浓度的死亡率的比例，按下列公式计算: （男S / N）* 100 其中: M S是死了个人的平均人数在分析样品 N是暴露的人的总数表1报告的暴露，以十二烷基硫酸钠（14D）与卤虫的测试数据的一个例子。 <table border="1"><tbody><tr><td> 浓度（毫克/升） </td>"&gt; 在每个副本中死亡的个体数量 </td><td> 死亡率（％） </td></tr><tr><td></td><td> 我 </td><td> 二 </td><td> 三 </td><td></td></tr><tr><td> 阴性对照 </td><td> 0 </td><td> 2 </td><td> 1 </td><td> 10 </td></tr><tr><td> 1.56 </td><td> 1 </td><td> 2 </td><td> 0 </td><td> 10 </td></tr><tr><td> 3.12 </td><td> 2 </td><td> 0 </td><td> 2 </td><td> 13 </td></tr><tr><td> 6.25 </td><td> 3 </td><td> 2 </td><td> 3 </td><td> 27 </td></tr><tr><td> 12.5 </td><td> 8 </td><td> 9 </td><td> 10 </td><td> 90 </td></tr><tr><td> 25.0 </td><td> 10 </td><td> 10 </td><td> 10 </td><td> 100 </td></tr><tr><td> 50.0 </td><td> 10 </td><td> 10 </td><td> 10 </td><td> 100 </td></tr></tbody></table>此外，确定LC 50值由一个图形高斯对数图，或使用适当的统计方法（例如斯皮尔曼，寇式的Probit方法）。表1的数据计算的LC 50为8.18（7.15-9.37）毫克/升。如果最高浓度测试造成的死亡率降低50％，你不应该进行，这将是不可靠的，甚至非可计算的LC 50值计算。然而，在这种情况下，将适当延长测试浓度范围内重复测试。另外，更正确的LC 50值表示为大于测试的最高浓度，可能表明，在测试的最高浓度的百分比死亡率和相应的没有效果的最高浓度。 结果被认为是有效的，如果在测试下列条件已得到满足: <ol class="lalpha"><li>控制的平均死亡率≤20％; </li><li>其中包括在14天内使用十二烷基硫酸钠，立法会50在8.0（±5）mg / L的时间间隔。 </li></ol>如果上述条件没有得到满足，与生物体的同一批获得的所有数据应视为无效，反复测试。

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没有利益冲突的声明。

卤虫是迄今所做的生态毒性测试和研究提供最有价值的测试生物体之一，让我们来说明它是可以维持有关卤虫使用毒理学和生态毒理学的几个选项。这个有机体变成一个合适的物种生物测定的特点是:广泛的地理分布，恶劣的环境条件和不同的营养素，相对简单的实验室培养和维护，抗操纵，生命周期短，大型苗种生产和高适应性大量存在的一些物种的信息。反对使用卤虫的批评被称为其灵敏度化学品的接触:早期的研究是指为较不敏感的物种的生态毒性研究卤虫 （暴露试验，以24小时），当其他测试生物相比，在相同的实验条件。的选择生态毒性测试方法是在这方面是至关重要的。我们想，规范了同一起跑线条件的协议曝光4至24小时（无节幼体作为测试生物，从使用囊肿）的方法，但相对较长的曝光时间（14天）可以提供更灵敏的反应。这个协议肯定可以比急性毒性试验更敏感的反应，但它不能被视为一种慢性试验的替代品，因为14天的接触是不相关的，如果我们考虑卤虫的估计寿命。端点选择此方法已被广泛的讨论。最初死亡率和增长（即14天的暴露后的头胸甲长）被选中，因为我们还希望确保一个长期的测试的亚致死效应的观察。然而，亚致死终点被认为是不太敏感相比，死亡率6。出于这个原因的死亡率是唯一的端点中提到的不伦不类离子的协议和视频。这一发现与其他研究人员的研究7,8观察，生存是最敏感的端点之间那些认为他们（生存，生长和繁殖）是一致的。 该方法是有用的评估毒性化学品，废水和环境矩阵9。在功能，它可能是有趣的测试其他亚致死的慢性毒性作用，如生物标志物（即酶的活性）10。

Loredana Manfra环境的保护和研究研究所（伊斯普拉部环境质量监测）区域环境保护署（艾米利亚 - 罗马涅，意大利）和Federica Savorelli产生。 海水/苦咸水和水质的UNICHIM委员会（在化工行业的技术标准化工作的机构）的沉积物- 卤虫甲壳类生物方法集团内的子工作组议员参考。 录像和编辑的Marco，伊斯普拉（科学咨询和文件服务）Pisapia。 由安娜·玛丽亚·西塞罗和Erika伊斯普拉Magaletti的的环境质量监测部生产协调。 我们想感谢艾米利亚 - 罗马涅环境保护区域代理，费拉拉科及BiologiCal的方法集团 - 海水/苦咸水和泥​​沙的UNICHIM委员会的合作水质;洛舍Boscolo和马西莫加贝里尼的，伊斯普拉（预防和减轻影响）的财政支持;和伦敦在罗马的语言学校他们的语言支持。 <table border="1"><tbody><tr><td> 试剂名称 </td><td> 公司 </td><td> 产品编号 </td></tr><tr><td>十二烷基硫酸钠</td><td> Sigma-Aldrich公司</td><td> L4390 </td></tr></tbody></table>附录藻媒体必须准备使用分析质量试剂。 一公升营养液溶解300纳米，30 KH 2 PO 4和20的NH 4氯蒸馏水ĞĞĞ准备。 四（甲，乙，丙，丁的初步解决方案矿物质）准备如下: <ul><li> ""一公升溶液含30一水硫酸锌4个H 2 O，25克， 硫酸铜 5 H 2 O，COSO 4 7 H 2 O 30克，硫酸锰4 H 2 O在蒸馏水和20ĞĞ; </li><li>升蒸馏水中含有50克的氯化铁3•H 2 O"B"的一个解决方案; </li><li>一公升的解决方案的"C"的Na 2 MOO 4•2 H 2 O在蒸馏水中含有25克; </li><li>一公升的解决方案的"D"中含有50克DI的Na 2 EDTA•2 H ​​2 O蒸馏水。 </li></ul>一公升的矿物质（解决方案的"E"）的最终解决方案，得到上述解决方案，以蒸馏水加入等分。除下列顺序建议:解决方案的"D"100毫升，10毫升的解决方案的"C"，"B"的解决方案和10毫升，10毫升的解决方案""。 三举措升解决方案的维生素（F，G，H）的准备如下: <ul><li>升蒸馏水中含有100毫克生物素的"F"的解决方案; </li><li>一个解决方案的"G"，含有10克蒸馏水硫胺素升; </li><li>一升蒸馏水中含有100毫克的钴胺素的"H"的解决方案。 </li></ul>一公升的维生素（"我"的解决方案）的最终解决方案，得到的三个初步方案中加入10毫升蒸馏水。 一公升的藻类媒体准备溶解的营养液，矿物的最终解决方案（解决方案的"E"）和在海水中的维生素A的最终解决方案（"我"的解决方案）1毫升1毫升1毫升。后者可能会获得公认的分析质量试剂市售配方或蒸馏水或去离子水溶解。市售创造了理想的海水盐混合物，例如即时海洋混合物。它是recommenDED与海水盐度30（±2）电源和温度（20±1）℃

long-term lethal toxicity test with the crustacean artemia franciscana

我们的研究活动的目标环境质量评价的生物学方法的使用，特别是海水/苦咸水和泥​​沙。生物指标的选择必须基于可靠的科学知识，可能的话，对标准化程序的可用性。在这篇文章中，我们提出了一个标准化的协议，用于海洋甲壳动物卤虫评估毒性化学品和/或海洋环境矩阵。科学家建议，丰年虾（ 卤虫 ）是一个合适的人选标准为全球利用生物测定的发展。已发表 ​​的论文数量，丰年虾（ 卤虫 ）的各种化学品和毒物的毒性作用。这种毒性研究的甲壳类动物的主要优势是干囊肿的整体可用性，这些都可以立即在测试中使用和培养困难不要求<suP&gt; 1,2。囊肿的毒性检测价格便宜，连续可用，简单，可靠，因此，一个重要的答案毒性筛选的常规需求，工业监测要求或监管目的3。该方法涉及作为端点死亡率。幸存者的数量进行了清点和死亡的百分比计算。幼虫被认为是死亡，如果他们没有表现出任何内部或外部的运动，在几秒钟的观察4。这个过程是标准化测试参考物质（十二烷基硫酸钠）;在这项工作中的一些结果报告。本文附带的视频描述程序的毒性测试的性能，显示所有的有关协议的步骤。

Watch this Scientific Journal Video about Long-term Lethal Toxicity Test with the Crustacean Artemia franciscana at JoVE.com

Long-term Lethal Toxicity Test with the Crustacean Artemia franciscana

这项研究涉及了宝贵的方法论协议，以确定长期（14天）致死毒性化学物质，工业废水或污水和海水甲壳类的液体环境样品施加的发展和标准化，<em&gt;卤虫franciscana</em&gt;。

致命的甲壳类动物长期毒性试验卤虫franciscana</em

Our research activities target the use of biological methods for the evaluation of environmental quality, with particular reference to saltwater/brackish ...

long-term-lethal-toxicity-test-with-the-crustacean-artemia-franciscana

Nanyang Technological University

Research

JoVE Journal

Biology

Long-term Lethal Toxicity Test with the Crustacean Artemia franciscana

19.5K 次观看.  Institute for Environmental Protection and Research. 这项研究涉及了宝贵的方法论协议，以确定长期（14天）致死毒性化学物质，工业废水或污水和海水甲壳类的液体环境样品施加的发展和标准化，卤虫franciscana。

视频: Long-term Lethal Toxicity Test with the Crustacean Artemia franciscana

Long-term Imaging Mammalian Cells using Wide-Field Microscopy

Actually what I'm gonna do, mount it in here first and then swap out You. So now I'm gonna aspirate try and be as gentle as possible.Okay. A few seconds just holding.Okay.Alright.So the important features of our lifestyle imaging grid are that, so it's an inverted scope, which means the objective, the samples here in the objective is underneath. So the objective is pointing up and the whole scope, or practically the whole, practically the whole scope, including the objectives and the sample are at 37 degrees. So everything in this plastic box is at 37 degrees.And right on top of the stage we have an air and CO2 mix. That is 5%CO2. So the idea is that the sample is actually in a cell, it's like a cell incubator.And so we have, this is our mixer for the air and the CO2 and, and it gets humidified and heated and then piped onto the, the sample on the stage.Okay. And today we're just gonna be doing fluorescence one channel GFP fluorescence. This is the hose that the CO2 and 5%CO2 goes in and it just fits into the stage holder.That's important. And then we can close these, take these down, and then we go to our first position. And what I'm gonna do is go to the first field, which is my control sample, and I'll pick positions based on what I see on the screen.So I just make sure I actually look into the microscope in each new, well, because the focal plane for each of the wells is different, the, the dish is not perfectly flat on the bottom. So you will need to make fine adjustments when you move from well to the well. So right now I'm on the first, well, what I'll do is I'll focus, so I just hit, I just told, I just turned on the light, the transmitted light for the microscope.And I have make sure all the light is going to the eye pieces. And so I just look in, make sure I get a nice clean field. And then what I'll do is turn that light off, turn the fluorescence light on, make sure the cells that I'm looking at look healthy and they're not apoptotic.And, and I turn that off. And what I do next is send, so now I'm gonna start picking positions, which means I have to te send the light path to the camera. This is the camera right here.So I tell the microscope to send all the camera, they don't see anything in the eye pieces. What I'll do is in the software controlling the microscope, I'll choose the right filter set, which for our purposes is psy. And then set an exposure time.And I know these cells very well and I know that they take a 0.2 second exposure time. So I tell, that's what I tell the software to do. And what I'll do is hit focus.This is, and it's basically just alive. The, A light will turn on and you can, oh, I don't know if you can see cells, but the reason this is a good field is because there's, all the cells are, even in terms of their fluorescence, There are no apoptotic cells. Apoptotic cells tend to be very bright and there's no dirt or schmutz in the field.And you can see that they're actually, this is a metaphase cell and that's a metaphase cell, that's a cell that's probably just exited mitosis. And I can just tell that because it looks like those are, those two sets of chromatin are very nicely oriented to each other. Like they just exited an face.So that's a good field. So, and now that I know that the exposure time and the filter set is correct, and this is gonna be software specific, I'll go and I'll take positions and now the software has memorized the X, Y, Z position of that field. And then when I do pick another field, it's, and I'll, so when I'm picking, when I'm picking new fields, but in the same, well, I'll use the, I'll move the i'll, I'll turn the shutter, I'll open the shutter and I'll look to see what I see on the screen and I'll pick fields based on that versus looking in the field.So I don't see any of my tonics in there. But again, even nicely centered, these cells will go through my PS I continue, here's a good example. Here's a good example of what I would avoid that, see how bright that is?That's very bright. That's probably remnants of an apoptotic cell. And the reason I would avoid that is because since it's so bright, when the software auto focuses, it will autofocus just on that and it won't be in the same plane as all these other cells.And so I won't get any information from that field. That looks nice. There's a, this is a pro metaphase right here, That's A metaphase.So, and I'm gonna take three positions per well. So that was three positions and now I'm going to go to my next Well, so you can, so what I will probably do is take images every 10 minutes. So it's a compromise.Lifestyle imaging is always a compromise, right? You have to image, you have to keep the cells healthy, especially if you're interested in mitosis. So you have to minimize their exposure to the fluorescent line.And, but you wanna get information. So you have to take images, you have to, so you have to compromise, you have to make a choice about what you want to capture, what kind of information you wanna capture. For our purposes, mitosis tends to take about an hour.So if we're taking 10 minute intervals, that's pretty good. We see prophase, we see metaphase, we see anaphase, and we can, we can see when there are, there are treatments or drugs or siRNAs that affect those specific cell cycle stages. So for our purposes, that's enough, but we always want more.We always want more positions or finer time and interval or something like that. So it's a compromise. So, and then I'll hit start.And so our software, it does an autofocusing pass on the first pass. So once I hit start, it should start auto autofocusing and you'll see the shutter open, open and close between auto focusing. So I'll hit start.So that's the shutter opening and closing. The Z motor is engaging and it's actually trying to find the plane with the most contrast, which will be the most in focus. You can actually see on the screen going in and out of finding the best plane of focus that might be worrisome.So what it does is it, it drives the Z motor to find the best, the most in focus plane focus, well the most in image. And then it acquires picture there and then it learns that Z position. And then we'll use that Z position for the next 12 passes.And then we'll do the auto focusing pass again. What Kind of experiments do they do? Okay, so our lab is mostly interested in understanding what regulates mitic progression, or at least I'm most interested in understanding that.So with live cell imaging and especially our multi position, long-term imaging scope, you can take, you can film cells for two or three days entering anding mitosis, and you can image multiple rounds of mitosis so you can understand. And then you can put drugs or RNAs or any kind of perturbations on the cells that you would like to study. So you can understand how, how those drugs or, or genes are, are important in regulating mitotic progression.Because it's not fixed cell work and live cell work, you can understand what, when you need to have a perturbation to affect mitosis or what stage of mitosis is affected. So it's really powerful in that respect. What, what are the main technical elements of this experiments?What are the possible problems? Well, the biggest problem besides getting the rig set up, getting the rig set up is not trivial. A lot of people have the rig, but just making sure it works well for your application.The financial element is not, you know, is not small. So there's the setting up of the apparatus and then, and then once you gather the data, there's a real data analysis issue. It takes these movie, if, so, if you're gathering 40 movies each with 30 cells, so you have 40 in 40 fields that you just filmed and each of those fields has 30 cells, then you have to sit down with each one of those movies and look at each one of those cells in that movie.So it takes a really long time to do data analysis. And that's, so it takes a week to analyze data that it took you two days to collect. So there's a, that's a very big obstacle in terms of gathering lots of data.So it's not, while it's high throughput data collection, it's not high throughput analysis. So what would you make to make this experiment really high throughput? So our lab is in the process of developing a program that does the data analysis automatically.And it's a pretty decent program. It's not perfect. The human eye is always better, but it's getting there.So What are the interesting application of this experiment? Maybe other fields you can mention? Besides cell division?You mean for the, oh, for the live cell imaging? Yes.Well, you can do cell motility assays. You can, depending on the, the microscopy that's involved, you can, you know, look at how cells, you could look at tumor development if you really wanted to.But there, you, you're, if as long as you have the right imaging set up, you can, you're pretty much not limited in terms of anything. Our scope happens to be just a wide field and we use 20 by 20 x objective. So it's a pretty low resolution set up, but that's sufficient for what we need to see.So there are, but there definitely aren't, you wouldn't be limited. Anything that has any dynamic kind of experiment, which is biology, it would be, would really benefit from lifestyle imaging. So calcium imaging, although you couldn't do it on our apparatus, that's a live cell.You definitely need live cell imaging for that. Like I said, patient assays, motility assays, those kinds of things would really benefit from live cell imaging. Would you like to tell a few words about your previous experience?How long do you work on these techniques? What do you you work on before? Wow.So in my PhD I didn't work.I did a lot of confocal microscopy, but no lifestyle imaging. And so during my postdoc I was, I helped Dr.King get this apparatus that, that we just used to set up these fragment, get that up and running. And, and so during my postdoc, I had a lot of experience with both widefield with TimeLapse imaging on our scope, which is the widefield and also TimeLapse imaging with confocal microscopy.And I would say that it's really powerful. You get a lot of information, but it's not easy to get working perfectly. So it really requires a lot of attention and a lot of attention to details at every little step of the process.And, and then you still have problems. So it's a tough application, but you can really get, if, if it's what you need to have to, to study your problem, your scientific problem, then it, it's invaluable in that respect, but it's tough. So find someone who knows what they're doing and use them.That's my advice. So good.

长期使用宽视场显微镜的成像哺乳动物细胞

科研

生物学

Long-term Culture of Human Breast Cancer Specimens and Their Analysis Using Optical Projection Tomography

Breast cancer is a leading cause of mortality in the Western world. It is well established that the spread of breast cancer, first locally and later distally, is a major factor in patient prognosis. Experimental systems of breast cancer rely on cell lines usually derived from primary tumours or pleural effusions. Two major obstacles hinder this research: (i) some known sub-types of breast cancers (notably poor prognosis luminal B tumours) are not represented within current line collections; (ii) the influence of the tumour microenvironment is not usually taken into account.
We demonstrate a technique to culture primary breast cancer specimens of all sub-types. This is achieved by using three-dimensional (3D) culture system in which small pieces of tumour are embedded in soft rat collagen I cushions. Within 2-3 weeks, the tumour cells spread into the collagen and form various structures similar to those observed in human tumours1. Viable adipocytes, epithelial cells and fibroblasts within the original core were evident on histology. Malignant epithelial cells with squamoid morphology were demonstrated invading into the surrounding collagen. Nuclear pleomorphism was evident within these cells, along with mitotic figures and apoptotic bodies.
We have employed Optical Projection Tomography (OPT), a 3D imaging technology, in order to quantify the extent of tumour spread in culture. We have used OPT to measure the bulk volume of the tumour culture, a parameter routinely measured during the neo-adjuvant treatment of breast cancer patients to assess response to drug therapy. 
Here, we present an opportunity to culture human breast tumours without sub-type bias and quantify the spread of those ex vivo. This method could be used in the future to quantify drug sensitivity in original tumour. This may provide a more predictive model than currently used cell lines.

We have developed a collagen-based in vitro assay which promotes proliferation and invasion from samples of all breast cancer subtypes. Optical Projection Tomography, a three dimensional microscopy technique was utilised to visualise and quantify tumour expansion. This assay may be used to quantify drug response of individual tumour samples.

长期培养的人类乳腺癌标本，并利用光学投影层析成像分析

Breast cancer is a leading cause of mortality in the Western world. It is well established that the spread of breast cancer, first locally and later ...

Long-term, High-resolution Confocal Time Lapse Imaging of Arabidopsis Cotyledon Epidermis during Germination

Imaging in vivo dynamics of cellular behavior throughout a developmental sequence can be a powerful technique for understanding the mechanics of tissue patterning. During animal development, key cell proliferation and patterning events occur very quickly. For instance, in Caenorhabditis elegans all cell divisions required for the larval body plan are completed within six hours after fertilization, with seven mitotic cycles1; the sixteen or more mitoses of Drosophila embryogenesis occur in less than 24 hr2. In contrast, cell divisions during plant development are slow, typically on the order of a day 3,4,5 . This imposes a unique challenge and a need for long-term live imaging for documenting dynamic behaviors of cell division and differentiation events during plant organogenesis. Arabidopsis epidermis is an excellent model system for investigating signaling, cell fate, and development in plants. In the cotyledon, this tissue consists of air- and water-resistant pavement cells interspersed with evenly distributed stomata, valves that open and close to control gas exchange and water loss. Proper spacing of these stomata is critical to their function, and their development follows a sequence of asymmetric division and cell differentiation steps to produce the organized epidermis (Fig. 1).
This protocol allows observation of cells and proteins in the epidermis over several days of development. This time frame enables precise documentation of stem-cell divisions and differentiation of epidermal cells, including stomata and epidermal pavement cells. Fluorescent proteins can be fused to proteins of interest to assess their dynamics during cell division and differentiation processes. This technique allows us to understand the localization of a novel protein, POLAR6, during the proliferation stage of stomatal-lineage cells in the Arabidopsis cotyledon epidermis, where it is expressed in cells preceding asymmetric division events and moves to a characteristic area of the cell cortex shortly before division occurs. Images can be registered and streamlined video easily produced using public domain software to visualize dynamic protein localization and cell types as they change over time.

Long-term, High-resolution Confocal Time Lapse Imaging of Arabidopsis Cotyledon Epidermis during Germination

We describe a protocol using chamber slides and media to immobilize plant cotyledons for confocal imaging of the epidermis over several days of development, documenting stomatal differentiation. Fluorophore-tagged proteins can be tracked dynamically by expression and subcellular localization, increasing understanding of their possible roles during cell division and cell-type differentiation.

从长期来看，高解析度激光共聚焦时间流逝成像<em&gt;拟南芥子叶</em&gt;表皮萌发过程中

Imaging in vivo dynamics of cellular behavior throughout a developmental sequence can be a powerful technique for understanding the mechanics of tissue ...

Serial Enrichment of Spermatogonial Stem and Progenitor Cells (SSCs) in Culture for Derivation of Long-term Adult Mouse SSC Lines

Spermatogonial stem and progenitor cells (SSCs) of the testis represent a classic example of adult mammalian stem cells and preserve fertility for nearly the lifetime of the animal. While the precise mechanisms that govern self-renewal and differentiation in vivo are challenging to study, various systems have been developed previously to propagate murine SSCs in vitro using a combination of specialized culture media and feeder cells1-3.
Most in vitro forays into the biology of SSCs have derived cell lines from neonates, possibly due to the difficulty in obtaining adult cell lines4. However, the testis continues to mature up until ~5 weeks of age in most mouse strains. In the early post-natal period, dramatic changes occur in the architecture of the testis and in the biology of both somatic and spermatogenic cells, including alterations in expression levels of numerous stem cell-related genes. Therefore, neonatally-derived SSC lines may not fully recapitulate the biology of adult SSCs that persist after the adult testis has reached a steady state.
Several factors have hindered the production of adult SSC lines historically. First, the proportion of functional stem cells may decrease during adulthood, either due to intrinsic or extrinsic factors5,6. Furthermore, as with other adult stem cells, it has been difficult to enrich SSCs sufficiently from total adult testicular cells without using a combination of immunoselection or other sorting strategies7. Commonly employed strategies include the use of cryptorchid mice as a source of donor cells due to a higher ratio of stem cells to other cell types8. Based on the hypothesis that removal of somatic cells from the initial culture disrupts interactions with the stem cell niche that are essential for SSC survival, we previously developed methods to derive adult lines that do not require immunoselection or cryptorchid donors but rather employ serial enrichment of SSCs in culture, referred to hereafter as SESC2,3.
The method described below entails a simple procedure for deriving adult SSC lines by dissociating adult donor seminiferous tubules, followed by plating of cells on feeders comprised of a testicular stromal cell line (JK1)3. Through serial passaging, strongly adherent, contaminating non-germ cells are depleted from the culture with concomitant enrichment of SSCs. Cultures produced in this manner contain a mixture of spermatogonia at different stages of differentiation, which contain SSCs, based on long-term self renewal capability. The crux of the SESC method is that it enables SSCs to make the difficult transition from self-renewal in vivo to long-term self-renewal in vitro in a radically different microenvironment, produces long-term SSC lines, free of contaminating somatic cells, and thereby enables subsequent experimental manipulation of SSCs.

Serial Enrichment of Spermatogonial Stem and Progenitor Cells SSCs in Culture for Derivation of Long-term Adult Mouse SSC Lines

A simple method to derive and maintain spermatogonial stem and progenitor cell lines from adult mice is presented here. The method utilizes feeder cells originating from the somatic cell compartment of the adult mouse testis. This technique is applicable to common mouse strains, including transgenic, knock-out, and knock-in mice.

在文化序列富集的精原干细胞和祖细胞（SSCS）推导长期成年小鼠SSC线

Spermatogonial stem and progenitor cells (SSCs) of the testis represent a classic example of adult mammalian stem cells and preserve fertility for nearly ...

Ablation of a Single Cell From Eight-cell Embryos of the Amphipod Crustacean Parhyale hawaiensis

The amphipod Parhyale hawaiensis is a small crustacean found in intertidal marine habitats worldwide. Over the past decade, Parhyale has emerged as a promising model organism for laboratory studies of development, providing a useful outgroup comparison to the well studied arthropod model organism Drosophila melanogaster. In contrast to the syncytial cleavages of Drosophila, the early cleavages of Parhyale are holoblastic. Fate mapping using tracer dyes injected into early blastomeres have shown that all three germ layers and the germ line are established by the eight-cell stage. At this stage, three blastomeres are fated to give rise to the ectoderm, three are fated to give rise to the mesoderm, and the remaining two blastomeres are the precursors of the endoderm and germ line respectively. However, blastomere ablation experiments have shown that Parhyale embryos also possess significant regulatory capabilities, such that the fates of blastomeres ablated at the eight-cell stage can be taken over by the descendants of some of the remaining blastomeres. Blastomere ablation has previously been described by one of two methods: injection and subsequent activation of phototoxic dyes or manual ablation. However, photoablation kills blastomeres but does not remove the dead cell body from the embryo. Complete physical removal of specific blastomeres may therefore be a preferred method of ablation for some applications. Here we present a protocol for manual removal of single blastomeres from the eight-cell stage of Parhyale embryos, illustrating the instruments and manual procedures necessary for complete removal of the cell body while keeping the remaining blastomeres alive and intact. This protocol can be applied to any Parhyale cell at the eight-cell stage, or to blastomeres of other early cleavage stages. In addition, in principle this protocol could be applicable to early cleavage stage embryos of other holoblastically cleaving marine invertebrates.

Ablation of a Single Cell From Eight-cell Embryos of the Amphipod Crustacean Parhyale hawaiensis

The amphipod Parhyale hawaiensis is a promising model organism for studies of crustacean embryology and comparative arthropod development and evolution. This protocol describes a method for manual removal of single blastomeres from early cleavage stage embryos of Parhyale.

一个单细胞消融从甲壳动物端足八细胞胚胎<em&gt; Parhyale hawaiensis</em

The amphipod Parhyale hawaiensis is a small crustacean found in intertidal marine habitats worldwide. Over the past decade, Parhyale has emerged as a ...

Multilayer Mounting for Long-term Light Sheet Microscopy of Zebrafish

Light sheet microscopy is the ideal imaging technique to study zebrafish embryonic development. Due to minimal photo-toxicity and bleaching, it is particularly suited for long-term time-lapse imaging over many hours up to several days. However, an appropriate sample mounting strategy is needed that offers both confinement and normal development of the sample. Multilayer mounting, a new embedding technique using low-concentration agarose in optically clear tubes, now overcomes this limitation and unleashes the full potential of light sheet microscopy for real-time developmental biology.

The development of zebrafish can be followed over days with light sheet microscopy when embryos are embedded in optically clear polymer tubes with low-concentration agarose.

多层安装于斑马鱼的长期光表镜

Light sheet microscopy is the ideal imaging technique to study zebrafish embryonic development. Due to minimal photo-toxicity and bleaching, it is ...

LarvaSPA, A Method for Mounting Drosophila Larva for Long-Term Time-Lapse Imaging

Live imaging is a valuable approach for investigating cell biology questions. The Drosophila larva is particularly suited for in vivo live imaging because the larval body wall and most internal organs are transparent. However, continuous live imaging of intact Drosophila larvae for longer than 30 min has been challenging because it is difficult to noninvasively immobilizeimmobilizing larvae for a long time. Here we present a larval mounting method called LarvaSPA that allows for continuous imaging of live Drosophila larvae with high temporal and spatial resolution for longer than 10 hours. This method involves partially attaching larvae to the coverslip using a UV-reactive glue and additionally restraining larval movement using a polydimethylsiloxane (PDMS) block. This method is compatible with larvae at developmental stages from second instar to wandering third instar. We demonstrate applications of this method in studying dynamic processes of Drosophila somatosensory neurons, including dendrite growth and injury-induced dendrite degeneration. This method can also be applied to study many other cellular processes that happen near the larval body wall.

LarvaSPA, A Method for Mounting Drosophila Larva for Long-Term Time-Lapse Imaging

This protocol describes a method for mounting Drosophila larvae to achieve longer than 10 h of uninterrupted time-lapse imaging in intact live animals. This method can be used to image many biological processes close to the larval body wall.

LarvaSPA，一种用于长期延时成像的安装果蝇幼虫的方法

Live imaging is a valuable approach for investigating cell biology questions. The Drosophila larva is particularly suited for in vivo live imaging because ...

Isolation, Transfection, and Long-Term Culture of Adult Mouse and Rat Cardiomyocytes

Ex vivo culture of the adult mammalian cardiomyocytes (CMs) presents the most relevant experimental system for the in vitro study of cardiac biology. Adult mammalian CMs are terminally differentiated cells with minimal proliferative capacity. The post-mitotic state of adult CMs not only restricts cardiomyocyte cell cycle progression but also limits the efficient culture of CMs. Moreover, the long-term culture of adult CMs is necessary for many studies, such as CM proliferation and analysis of gene expression.
The mouse and the rat are the two most preferred laboratory animals to be used for cardiomyocyte isolation. While the long-term culture of rat CMs is possible, adult mouse CMs are susceptible to death and cannot be cultured more than five days under normal conditions. Therefore, there is a critical need to optimize the cell isolation and long-term culture protocol for adult murine CMs. With this modified protocol, it is possible to successfully isolate and culture both adult mouse and rat CMs for more than 20 days. Moreover, the siRNA transfection efficiency of isolated CM is significantly increased compared to previous reports. For adult mouse CM isolation, the Langendorff perfusion method is utilized with an optimal enzyme solution and sufficient time for complete extracellular matrix dissociation. In order to obtain pure ventricular CMs, both atria were dissected and discarded before proceeding with the disassociation and plating. Cells were dispersed on a laminin coated plate, which allowed for efficient and rapid attachment. CMs were allowed to settle for 4-6 h before siRNA transfection. Culture media was refreshed every 24 h for 20 days, and subsequently, CMs were fixed and stained for cardiac-specific markers such as Troponin and markers of cell cycle such as KI67.

Here, we present a protocol for the isolation, transfection, and long-term culture of adult mouse and rat cardiomyocytes.

成年小鼠和大鼠心肌细胞的隔离、转染和长期培养

Ex vivo culture of the adult mammalian cardiomyocytes (CMs) presents the most relevant experimental system for the in vitro study of cardiac biology. ...

Application of Passive Head Motion to Generate Defined Accelerations at the Heads of Rodents

Exercise is widely recognized as effective for various diseases and physical disorders, including those related to brain dysfunction. However, molecular mechanisms behind the beneficial effects of exercise are poorly understood. Many physical workouts, particularly those classified as aerobic exercises such as jogging and walking, produce impulsive forces at the time of foot contact with the ground. Therefore, it was speculated that mechanical impact might be implicated in how exercise contributes to organismal homeostasis. For testing this hypothesis on the brain, a custom-designed ''passive head motion'' (hereafter referred to as PHM) system was developed that can generate vertical accelerations with controlled and defined magnitudes and modes and reproduce mechanical stimulation that might be applied to the heads of rodents during treadmill running at moderate velocities, a typical intervention to test the effects of exercise in animals. By using this system, it was demonstrated that PHM recapitulates the serotonin (5-hydroxytryptamine, hereafter referred to as 5-HT) receptor subtype 2A (5-HT2A) signaling in the prefrontal cortex (PFC) neurons of mice. This work provides detailed protocols for applying PHM and measuring its resultant mechanical accelerations at rodents' heads.

The present protocol describes a custom-designed ''passive head motion'' system, which reproduces mechanical accelerations at rodents' heads generated during their treadmill running at moderate velocities. It allows dissecting mechanical factors/elements from the beneficial effects of physical exercise.

应用被动头部运动在啮齿动物头部产生定义的加速度

Exercise is widely recognized as effective for various diseases and physical disorders, including those related to brain dysfunction. However, molecular ...

Collection and Long-Term Maintenance of Leaf-Cutting Ants (Atta) in Laboratory Conditions

Ants are one of the most biodiverse groups of animals on the planet and inhabit different environments. The maintenance of ant colonies in controlled environments enables an enriched comprehension of their biology that can contribute to applied research. This practice is usually employed in population control studies of species that cause economic loss, such as Atta ants. To cultivate their mutualistic fungus, these leaf-cutting ants collect leaves and for this are considered agricultural pests widely distributed throughout the American continent. They are highly socially organized and inhabit elaborated underground nests composed of a variety of chambers. Their maintenance in a controlled environment depends on a daily routine of several procedures and frequent care that are described here. It starts with the collection of queens during the reproductive season (i.e., nuptial flight), which are then individually transferred to plastic containers. Due to the high mortality rate of queens, a second collection can be carried out about 6 months after the nuptial flight, when incipient nests with developed fungus wad are excavated, hand-picked, and placed in plastic containers. In the laboratory, leaves are daily provided to established colonies, and ant-produced waste is weekly removed along with remaining dry plant material. As the fungus garden keeps growing, colonies are transferred to different types of containers according to the experimental purpose. Leaf-cutting ant colonies are placed in interconnected containers, representing the organizational system with functional chambers built by those insects in nature. This setup is ideal to monitor factors such as waste amount, fungus garden health, and the behavior of workers and queen. Facilitated data collection and more detailed observations are considered the greatest advantage of keeping ant colonies in controlled conditions.

Collection and Long-Term Maintenance of Leaf-Cutting Ants Atta in Laboratory Conditions

Here, a protocol is described to successfully collect and maintain healthy Atta (Hymenoptera: Formicidae) ant colonies in laboratory conditions. Additionally, different nest types and configurations are detailed together with possible experimental procedures.

在实验室条件下收集和长期维护切叶蚁（Atta）

Ants are one of the most biodiverse groups of animals on the planet and inhabit different environments. The maintenance of ant colonies in controlled ...