1。协议利用细胞培养多保温瓶<ol><li> 船只，并加入细胞悬液的制备<ol><li>需要准备细胞的生长介质。 </li><li>行了多瓶，它面临在工作面上（无菌层流罩）第的一面垂直。 <ol><li> 这些烧瓶是在3和5层的格式，并使用类似的工作流模式作为一个T - 175烧瓶。 </li></ol></li><li>松开并取下帽子。添加到使用50或100毫升吸管瓶或浇需要预热中等量。 <ol><li> 滴管，≤10毫升即可到达容器底部时多保温瓶放在第朝上垂直。移液器≥10毫升立即到船只可以达到过去多瓶上的标志，从而提供了方便的门户媒体船只添加更大容量。 </li></ol></li><li>为了避免介质的冒泡，让液体流FLOW沿内壁的多瓶的盖子（徽标端）。 </li><li>通过前层用10 mL的吸管和第烧瓶从一个集中的细胞股票添加到生长介质细胞悬液。 提示: -运输多保温瓶上的孵化器网站的购物车和执行其余的步骤 。 -细胞接种密度不同的细胞类型，媒介和文化的持续时间需要而异 。 开始播种密度和标准的T - 175烧瓶中使用的介质卷和乘以3或5，取决于多瓶使用的格式。 </li></ol></li><li> 混合细胞 <ol><li> 混合的位置是:保持多瓶直立面对你的标志，反过来逆时针45 °的角度面对你与混合端口。 </li><li>保持在相同的角度，轻轻地从正面倾斜多瓶，直到液体（颈部远离你）在顶层水渠充分downwar通过混合端口的DS。枢轴上混合端口端。 </li><li>同样，轻轻摇动多瓶，从后向前（朝你的颈部），直到介质完全通过混合端口的底层朝上方的水渠。重复步骤1.2.2和1.2.3更多的时间来确保正确的混合。 </li><li>多瓶混合位置（步骤1.2.1），并继续执行步骤1.3 </li><li>此外，细胞悬液，可从外部多瓶准备细胞悬液，可以添加的船只使用吸管或温柔浇筑。 <ol><li> 混合端口允许船与媒体的细胞混合，无需外部，使大量的细胞悬液。 </li><li> 它还允许跨多瓶层层媒体均衡 </li></ol></li></ol></li><li> 流体平衡的 <ol><li>地方多瓶，混合后/加入细胞悬液，在平坦的工作表面垂直，以平衡液体体积方程LLY在所有的层。 </li></ol></li><li> 每一层的分区液体进入<ol><li>你面临的标志保持多瓶，并顺时针旋转45 °角进行分区到每一层液体。 提示: -为了达到最佳效果，重要的是平坦的工作表面使用的步骤1.3-1.4 </li></ol></li><li> 交通运输 <ol><li>一旦媒体含有细胞悬液被分割，运输多瓶相同的45 °角（顺时针方向）在与媒体远离进入孵化器的混合端口1.4.1步骤。从孵化器中删除时，烧瓶，在显微镜上观看，也适合这个位置。 </li></ol></li><li> 孵化器的货架放置到多瓶 <ol><li>控股多瓶，在45 °角（顺时针方向，远离混合端口），轻轻旋转，横向到孵化器的表面（从混合端口使用的角落里支点）。莱多瓶，标志朝上。 <ol><li> 猎鹰多瓶设计允许堆放的船只，并认为他们的地方在一个坚固的堆叠肋骨。 </li></ol></li></ol></li><li> 分布的细胞和试剂 <ol><li>多瓶平放在工作表面上后，轻轻摇动来回侧侧分发到文化的照顾，不要对泄漏液体从每一层表面的细胞均匀。堆栈烧瓶。 <ol><li> 这种药剂是由透明材料，可以很容易地在显微镜观看和细胞上的最后一层。 </li></ol></li></ol></li><li> 媒体交流 <ol><li>吸而倾斜多瓶到左边（混合港口边）你面临的标志。 </li><li>然后倾斜，多瓶的权利，继续吸出所有剩余媒体。 </li><li>添加适量的媒体，并按照步骤1.3-1.7 </li></ol></li></ol> 2。从多，保温瓶收获细胞<ol><li>游离细胞，最多多的保温瓶，垂直线，使每一个混合的位置（步骤1.2.1） 。轻轻地颠倒的脖子上，面临你，让媒体流失的顶层多瓶瓶。 </li><li>插入一个5年或10毫升通过颈部的吸尖，直到您到达液面附近的混合端口。吸用尽介质。 </li><li>添加解离试剂/洗涤缓冲液，使混合在步骤1.2.1 中的地位，然后进行后续步骤1.3-1.7中生长介质/中和剂倒入一个接收管细胞悬液或反转烧瓶等细胞流失容器顶部层和收集细胞，用10毫升吸管。 提示 :为了提高细胞或试剂的复苏，带来多瓶组合位置（步骤1.2.1），对运营商与颈部反转，让媒体人的完整的排水系统<html> L层到顶层。然后，倾斜多瓶顺时针45 °角（远离组合端口），而多瓶仍然倒。使用移液管（1-10毫升）收集任何其余试剂。 </li></ol> 3。推荐工作容积在猎鹰多瓶生长介质 3层:每多瓶75-150毫升 5层:每多瓶125-250毫升解离剂 3层:≥15毫升，每多瓶 5层:≥25毫升，每多瓶 提示:开始使用标准的T - 175烧瓶中等量，乘以3年或5倍多瓶的格式取决于评估，使每单位面积毫升保持不变 。 4。代表性的成果: 1 猎鹰多瓶的设计。 files/ftp_upload/3418/3418fig1.jpg"/&gt; 图1:多瓶细胞培养容器可在3 - 5层，提供525和875平方厘米表面生长区，分别的细胞规模可堆叠格式。移液器的访问有利于增加和清除细胞及试剂和出的船只。混合端口的存在允许快速船混合和跨多瓶的所有层媒体均等。 2， 细胞产量，使用多瓶: 这些船只在3层和5层的格式，对应于3和5倍的T - 175烧瓶表面积。因此，≥3倍和5倍（130 ± 6.8 × 10 6和218 ± 23.6 × 10 6细胞，分别）BHK - 21号（幼仓鼠肾细胞）种植，并从多瓶回收相比，T - 175瓶（43.2 ± 3.5 × 10 6细胞;图2a）。每U的细胞产量NIT表面面积相当于3 - 5层多瓶和BHK - 21的T - 175瓶中，前列腺癌细胞LNCaP（人类前列腺腺癌细胞株），对H​​ep - G2（人肝癌细胞系），ECOPACK 2-293（人肾细胞）培养期为48 - 96H（图2b）在生长介质（35毫升）每一层细胞供应商（ATCC，Sigma和/或Clontech公司）的建议。列举一个自动化的VI -细胞计数（1）细胞。 <img alt="图2A" src="/files/ftp_upload/3418/3418fig2A.jpg" /> 图2A:三到五倍的BHK - 21细胞生长和恢复3 - 5层多保温瓶相比，T - 175烧瓶。预期收益率是使用控制细胞平均产量确定的T - 175烧瓶三到五倍乘以3 - 5层多保温瓶（N = 4烧瓶/格式）。 <img alt="图2B" src="/files/ftp_upload/3418/3418fig2B.jpg" /><br/&gt; 图2B细胞每平方厘米2的产量相当于在3 - 5层多瓶和BHK - 21，肝癌，前列腺癌细胞LNCaP EcoPack2 - 293细胞的T - 175烧瓶。每个条形代表的平均4至6烧瓶。 BHK - 21细胞（11000 cells/cm2）培养72小时，LNCaP细胞（20000 cells/cm2）EcoPack2 - 293细胞（〜35,000 cells/cm2）培养96小时，HepG2细胞（25000细胞/厘米2 ）培养48小时前收获。 3， 媒体之间分配层多瓶 细胞培养液（DMEM; Invitrogen公司）被添加到5层多瓶（250 mL/5-layer船只）和成层根据协议，上面描述的分区。通过钻探孔，在每一层和泵从个人层次的媒体，媒体分发。从每一层的液体的重量是相对统一的层与层之间，如在图3所示。它们如下:510.8 ± 0.73，50.3 ± 0.58，50.21 ± 0.13 49.88 ± 0.35 49.45 ± 0.37（GM）。 <img alt="图3" src="/files/ftp_upload/3418/3418fig3.jpg" /> 图3。均匀介质分布在5层多瓶五层。培养液中添加了多瓶（250 ml/5-layer船），平衡及分割为单个图层。通过媒体被抽成单个层钻孔和液体的重量，从每一层（N = 6瓶）的记录。 多瓶层间细胞分布 4。 可以增加细胞内的多瓶混合。我们模拟的细胞之间的分配使用多瓶大小相似，对细胞的珠层（10微米; PolySciences公司）。珠悬液加入到多瓶使用10毫升吸管通过顶层的船只和船只为递减媒体混合ribed在上面的协议。珠分布在每一层液和含珠被抽暂停从个人层次的钻孔。珠浓度从每一层上库尔特计数器读和记录。下面显示的是等价的层间珠分布在3层多瓶（图4a）。混合端口，使细胞和试剂多瓶层之间的均匀分布。 <img alt="图4A" src="/files/ftp_upload/3418/3418fig4A.jpg" /> 图4a:珠分布在3层多瓶三层。暂停的珠子（3.6 × 10 6 /毫升）加入到多保温瓶免除中等（珠悬挂:介质体积为1:10，第二卷:第一卷）和混合型，平衡和分区步骤，使用的协议描述。阅读上的库尔特计数珠浓度从每一层（中等通过对每一层钻孔抽水）ER和记录。下面显示的是等效的层间珠分布在3 - 层多瓶（N = 5烧瓶） 所示ECOPACK 2-293增长3层补充生长介质中的多，保温瓶&gt; 80％汇合的细胞染色模式的代表图像。细胞单层固定和结晶紫染色和多瓶层，然后切和图像扫描（2）。注意，细胞图案多瓶（图4b）的所有层上的同质性。类似的结果，获得与评估（数据未显示）的多种细胞类型。 <img alt="图4B" src="/files/ftp_upload/3418/3418fig4B.jpg" /> 图4b:这个数字说明的多保温瓶层之间的同质化的细胞生长。 ECOPACK - 2 - 293细胞生长在3层多瓶和T - 175&gt; 80％汇合固定，结晶紫染色。多瓶血管被切断，每个染色层扫描。 5，多瓶的空气供应。 花EcoPack2 - 293细胞培养96小时使用BioProfile佛雷斯分析仪（3,4）（诺瓦生物医学）媒体分析，没有发现在5层多保温瓶与T - 175瓶（81.03细胞生长的空气饱和差± 1.9比83.4 ± 5.8％O 2的环境）。 <img alt="图5" src="/files/ftp_upload/3418/3418fig5.jpg" /> 图5:空气饱和度（％O 2的环境）花了媒体类似的预混合媒体从5层多瓶与T - 175烧瓶。 EcoPack2 - 293细胞接种在35,000个细胞/厘米2的密度和培养96小时之前，媒体分析（N = 3瓶） 。

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所有的作者是Becton Dickinson公司，生物科学分部，发现医学实验组的员工。

目前的研究表明，生产率的提高，多瓶的设计研究人员提供。虽然重要的是要按照上述最佳性能概述使用时多，保温瓶的步骤，有几个关键在这个协议中，被视为最重要的步骤。这些措施包括:（一）使用组合港口内的船只运送后分割成每个层层铺设多瓶平后，分区（三）流体顺时针45 °角度多瓶（二）细胞和试剂混合孵化器。 多瓶的正确使用，导致生产同质化的细胞群内每艘船的 T - 175瓶（5）是一致的环境中培养。这些船只提供类似的足迹细胞的T - 175烧瓶3倍和5倍以上。 3和5层的船只提供，分别为525和875厘米增长领域，并提供空间和劳动力小号用户avings。组织培养表面处理媲美从而而不需要规模的标准烧瓶重新优化现有的文化条件或牺牲质量，同质性或细胞（6,7）的性能。这也提供了与以前收集的数据的可比性。这些船只也可以涂上试剂，如胶原蛋白，纤维连接蛋白，D -聚赖氨酸的附着，生长和某些类型的细胞，如肝细胞8，kertainocytes 9，在无血清培养基中生长的干细胞分化为专门的底层配方。涂层解决方案，可清除微小残留药液保留，以减少浪费宝贵的试剂以及有效去除涂层解决方案之前培养细胞。建议最佳介质卷培养细胞在多瓶范围从0.142-0.287毫升/厘米2，这意味着每一层25-50毫升。不像另一个多层瓶，T他的产品提供快速船混合均匀分布的细胞内，每艘船的层间试剂以及在船只允许混合端口。不同的细胞系，小学文化和干细胞已被有效地扩大使用多的保温瓶。这些船只特别有利的高通量筛选，生产疫苗，病毒载体转染和细胞治疗的细胞，如大量的应用需求。 节省时间，空间，劳动力和减少废物的产生，是传统文化与单层船只多瓶的关键的奖金。我们可以约5中提取的干细胞数量的三倍，文化的T - 175烧瓶在同一空间使用3个，5层多保温瓶。这在节省空间的优点是不局限于为T - 175只，但也可以扩展到其他船只:一个标准滚瓶设备，适合常见的实验室孵化器房子〜4 ROLLER瓶（2200毫升），其中每个提供850厘米 2表面积。 〜20日，5层多保温瓶可以在同一地区，被安置，从而提供了5倍的增长表面细胞培养。此外，在"走出去绿色"意识的提高，减少废物的产生的选项是非常可取的的。在这方面，有一个38％（5，T - 175瓶中的重量，而之一，5层多瓶的重量〜400克〜640克）减少废弃物产生，使用多功能保温瓶相比，T - 175瓶和这些优势导致减少废物贮存，处置成本和节省用户的经济。

<table border="1"><tbody><tr><td> 试剂/设备名称 </td><td> 公司 </td><td> 目录＃ </td><td> 评论 </td></tr><tr><td> 3层组织培养处理525厘米2 </td><td> BD公司</td><td> 353143 </td><td rowspan="21"> <a href="http://www.bdbiosciences.com/cellculture/multiflasks/index.jsp">BD公司细胞培养- BD猎鹰多，烧瓶</a> </td></tr><tr><td> 5层的组织培养处理875厘米2 </td><td> BD公司</td><td> 353144 </td></tr><tr><td> 100毫升移液器</td><td> BD公司</td><td> 357600 </td></tr><tr><td> 10毫升吸管</td><td> BD公司</td><td> 357551 </td></tr><tr><td> 5毫升吸吸管</td><td> BD公司</td><td&gt; 357501 </td></tr><tr><td> 50毫升的聚丙烯锥形管</td><td> BD公司</td><td> 352070 </td></tr><tr><td> T - 175瓶组织培养处理</td><td> BD公司</td><td> 353028 </td></tr><tr><td>革兰氏结晶紫</td><td>屋宇署</td><td> 212525 </td></tr><tr><td> DMEM培养液</td><td> Invitrogen公司</td><td> 11885 </td></tr><tr><td> GMEM </td><td>西格玛</td><td> G5154 </td></tr><tr><td> RPMI - 1640 </td><td> ATCC </td><td> 30-2001 </td></tr><tr><td>纪念</td><td> ATCC </td><td> 30-2003 </td></tr><tr><td>胰蛋白酶EDTA </td><td>龙沙</td><td> CC - 5012 </td></tr><tr><td>胎牛血清</td><td> Invitrogen公司</td><td> 16000-044 </td></tr><tr><td> Tryptose磷酸盐肉汤</td><td>西格玛</td><td> T8159 </td&gt; </tr><tr><td> BHK - 21细胞</td><td>西格玛</td><td> 85011433 </td></tr><tr><td>对Hep - G2，前​​列腺癌细胞LNCaP </td><td> ATCC </td><td></td></tr><tr><td> ECOPACK 2-293 </td><td> Clontech公司</td><td></td></tr><tr><td>多聚甲醛</td><td>电子显微镜科学</td><td> 15710 </td></tr><tr><td>聚苯乙烯珠</td><td> PolySciences公司</td><td> 24628-20 </td></tr><tr><td>生物个人资料的Flex分析仪</td><td>诺瓦生物医学</td><td></td></tr></tbody></table>

scale-up of mammalian cell culture using a new multilayered flask

一个基于单元的应用越来越多，需要大量的细胞。单层T型瓶，是在小规模的扩大足够的使用，有可能成为累赘，费力又费时，需要大量的细胞。要解决这个问题需要，一个新的多层细胞培养容器的性能，以促进细胞单层的T -烧瓶易规模将讨论。烧瓶测试可在3 - 5层的格式和使文化和完全恢复三种细胞数量的五倍，比T - 175烧瓶。屋宇署多瓶的一个主要特点是混合/平衡端口，允许在血管混合以及内部和之间的每艘船舶层细胞和试剂的均匀分布，并不断产生的环境中，可以培养细胞快速是一致的T - 175烧瓶。 这些多保温瓶的设计也允许对C直接添加到瓶中的试剂和细胞以及高效回收有价值的细胞和试剂onvenient吸管访问和降低污染浇筑风险。浇筑超过吹打首选的应用程序，设计允许微小残留药液保留，以减少浪费宝贵的细胞和试剂。

Watch this Scientific Journal Video about Scale-Up of Mammalian Cell Culture using a New Multilayered Flask at JoVE.com

Scale-Up of Mammalian Cell Culture using a New Multilayered Flask

细胞发挥在研究的工具和越来越大的作用，并发现和开发新疗法。有了这个我们需要更有效率和更有效的方式，为种植和收获附件依赖细胞的细胞数量增加的需要。一个正确的功能的多层瓶中可以达到这个目的。

哺乳动物细胞培养，使用一种新的多层瓶中的比例增加

A growing number of cell-based applications require large numbers of cells. Usage of single layer T-flasks, that are adequate during small-scale ...

scale-up-of-mammalian-cell-culture-using-a-new-multilayered-flask

Research

JoVE Journal

Biology

37.5K 次观看.  BD Biosciences. 细胞发挥在研究的工具和越来越大的作用，并发现和开发新疗法。有了这个我们需要更有效率和更有效的方式，为种植和收获附件依赖细胞的细胞数量增加的需要。一个正确的功能的多层瓶中可以达到这个目的。

视频: Scale-Up of Mammalian Cell Culture using a New Multilayered Flask

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

Ole Isacson: Development of New Therapies for Parkinson's Disease

Ole Isacson gives a concise overview of Parkinsons's disease, its causes, therapeutic strategies, and advances in Parkinson's research.

OLE Isacson：帕金森氏病的新疗法的发展

Laser Capture Microdissection of Mammalian Tissue

Laser capture microscopy, also known as laser microdissection (LMD), enables the user to isolate small numbers of cells or tissues from frozen or formalin-fixed, paraffin-embedded tissue sections. LMD techniques rely on a thermo labile membrane placed either on top of, or underneath, the tissue section. In one method, focused laser energy is used to melt the membrane onto the underlying cells, which can then be lifted out of the tissue section. In the other, the laser energy vaporizes the foil along a path "drawn" on the tissue, allowing the selected cells to fall into a collection device. Each technique allows the selection of cells with a minimum resolution of several microns. DNA, RNA, protein, and lipid samples may be isolated and analyzed from micro-dissected samples. In this video, we demonstrate the use of the Leica AS-LMD laser microdissection instrument in seven segments, including an introduction to the principles of LMD, initializing the instrument for use, general considerations for sample preparation, mounting the specimen and setting up capture tubes, aligning the microscope, adjusting the capture controls, and capturing tissue specimens. Laser-capture micro-dissection enables the investigator to isolate samples of pure cell populations as small as a few cell-equivalents. This allows the analysis of cells of interest that are free of neighboring contaminants, which may confound experimental results.

激光捕获显微切割哺乳动物组织

Laser capture microscopy, also known as laser microdissection (LMD), enables the user to isolate small numbers of cells or tissues from frozen or ...

Development of New Therapeutic Applications Using Microfluidics

使用微流体开发新的治疗应用

Method for Culture of Early Chick Embryos ex vivo (New Culture)

The chick embryo is a valuable tool in the study of early embryonic development. Its transparency, accessibility and ease of manipulation, make it an ideal tool for studying  the formation and patterning of brain, neural tube, somite and heart primordia. Applications of chick embryo culture include electroporation of DNA or RNA constructs in order to analyze gene function, grafts of growth factor coated beads such as FGFs and BMPs , as well as whole mount in situ hybridization and immunohistochemistry. This video demonstrates the different steps in chick embryo culture; First, the embryo is explanted in saline. Then, the embryo is centered on a glass ring. The membranes surrounding the embryo are lifted along the walls of the ring. The ring is then placed in a culture dish containing a pool of albumine. The culture dish is sealed and placed in a humid chamber, where the embryo is cultured for up to 24 hrs. Finally, the embryo is removed from the ring, fixed and processed for further applications. A troubleshooting guide is also presented.

Method for Culture of Early Chick Embryos ex vivo New Culture

This video demonstrates New culture, a method by which chick embryos are cultured outside the egg for up to 24 hr. This method enables one to study early development (primitive streak to 14 som.), a period corresponding to E7-9 in mouse. Applications of this technique include electroporation, in situ hybridization and immunohistochemistry.

文化早期鸡胚体外（新文化）的方法

The chick embryo is a valuable tool in the study of early embryonic development. Its transparency, accessibility and ease of manipulation, make it an ...

Determining Cell Number During Cell Culture using the Scepter Cell Counter

Counting cells is often a necessary but tedious step for in vitro cell culture. Consistent cell concentrations ensure experimental reproducibility and accuracy. Cell counts are important for monitoring cell health and proliferation rate, assessing immortalization or transformation, seeding cells for subsequent experiments, transfection or infection, and preparing for cell-based assays. It is important that cell counts be accurate, consistent, and fast, particularly for quantitative measurements of cellular responses.
Despite this need for speed and accuracy in cell counting, 71% of 400 researchers surveyed1 who count cells using a hemocytometer. While hemocytometry is inexpensive, it is laborious and subject to user bias and misuse, which results in inaccurate counts. Hemocytometers are made of special optical glass on which cell suspensions are loaded in specified volumes and counted under a microscope. Sources of errors in hemocytometry include: uneven cell distribution in the sample, too many or too few cells in the sample, subjective decisions as to whether a given cell falls within the defined counting area, contamination of the hemocytometer, user-to-user variation, and variation of hemocytometer filling rate2.
To alleviate the tedium associated with manual counting, 29% of researchers count cells using automated cell counting devices; these include vision-based counters, systems that detect cells using the Coulter principle, or flow cytometry1. For most researchers, the main barrier to using an automated system is the price associated with these large benchtop instruments1.
The Scepter cell counter is an automated handheld device that offers the automation and accuracy of Coulter counting at a relatively low cost. The system employs the Coulter principle of impedance-based particle detection3 in a miniaturized format using a combination of analog and digital hardware for sensing, signal processing, data storage, and graphical display. The disposable tip is engineered with a microfabricated, cell- sensing zone that enables discrimination by cell size and cell volume at sub-micron and sub-picoliter resolution. Enhanced with precision liquid-handling channels and electronics, the Scepter cell counter reports cell population statistics graphically displayed as a histogram.

The Scepter Cell Counter is a handheld automated device that can be used to count cells, monitor cell diameter and volume, and be used to check the health and quality of cellular populations from one culture to the next.

确定在细胞培养的细胞数量，使用权杖细胞计数

Counting cells is often a necessary but tedious step for in vitro cell culture. Consistent cell concentrations ensure experimental reproducibility and ...

Analysis of Cell Cycle Position in Mammalian Cells

The regulation of cell proliferation is central to tissue morphogenesis during the development of multicellular organisms. Furthermore, loss of control of cell proliferation underlies the pathology of diseases like cancer. As such there is great need to be able to investigate cell proliferation and quantitate the proportion of cells in each phase of the cell cycle. It is also of vital importance to indistinguishably identify cells that are replicating their DNA within a larger population. Since a cell&prime;s decision to proliferate is made in the G1 phase immediately before initiating DNA synthesis and progressing through the rest of the cell cycle, detection of DNA synthesis at this stage allows for an unambiguous determination of the status of growth regulation in cell culture experiments.
DNA content in cells can be readily quantitated by flow cytometry of cells stained with propidium iodide, a fluorescent DNA intercalating dye. Similarly, active DNA synthesis can be quantitated by culturing cells in the presence of radioactive thymidine, harvesting the cells, and measuring the incorporation of radioactivity into an acid insoluble fraction. We have considerable expertise with cell cycle analysis and recommend a different approach. We Investigate cell proliferation using bromodeoxyuridine/fluorodeoxyuridine (abbreviated simply as BrdU) staining that detects the incorporation of these thymine analogs into recently synthesized DNA. Labeling and staining cells with BrdU, combined with total DNA staining by propidium iodide and analysis by flow cytometry1 offers the most accurate measure of cells in the various stages of the cell cycle. It is our preferred method because it combines the detection of active DNA synthesis, through antibody based staining of BrdU, with total DNA content from propidium iodide. This allows for the clear separation of cells in G1 from early S phase, or late S phase from G2/M. Furthermore, this approach can be utilized to investigate the effects of many different cell stimuli and pharmacologic agents on the regulation of progression through these different cell cycle phases.
In this report we describe methods for labeling and staining cultured cells, as well as their analysis by flow cytometry. We also include experimental examples of how this method can be used to measure the effects of growth inhibiting signals from cytokines such as TGF-&beta;1, and proliferative inhibitors such as the cyclin dependent kinase inhibitor, p27KIP1. We also include an alternate protocol that allows for the analysis of cell cycle position in a sub-population of cells within a larger culture5. In this case, we demonstrate how to detect a cell cycle arrest in cells transfected with the retinoblastoma gene even when greatly outnumbered by untransfected cells in the same culture. These examples illustrate the many ways that DNA staining and flow cytometry can be utilized and adapted to investigate fundamental questions of mammalian cell cycle control.

Determining the cell cycle position of a population of cells, or understanding how signals affect proliferation, can be readily measured by flow cytometry using this protocol. We report a simple experimental approach to staining cells and quantifying their position in the cell cycle.

在哺乳动物细胞中细胞周期位置分析

The regulation of cell proliferation is central to tissue morphogenesis during the development of multicellular organisms. Furthermore, loss of control of ...

Determination of Mammalian Cell Counts, Cell Size and Cell Health Using the Moxi Z Mini Automated Cell Counter

Particle and cell counting is used for a variety of applications including routine cell culture, hematological analysis, and industrial controls1-5. A critical breakthrough in cell/particle counting technologies was the development of the Coulter technique by Wallace Coulter over 50 years ago. The technique involves the application of an electric field across a micron-sized aperture and hydrodynamically focusing single particles through the aperture. The resulting occlusion of the aperture by the particles yields a measurable change in electric impedance that can be directly and precisely correlated to cell size/volume. The recognition of the approach as the benchmark in cell/particle counting stems from the extraordinary precision and accuracy of its particle sizing and counts, particularly as compared to manual and imaging based technologies (accuracies on the order of 98% for Coulter counters versus 75-80% for manual and vision-based systems). This can be attributed to the fact that, unlike imaging-based approaches to cell counting, the Coulter Technique makes a true three-dimensional (3-D) measurement of cells/particles which dramatically reduces count interference from debris and clustering by calculating precise volumetric information about the cells/particles. Overall this provides a means for enumerating and sizing cells in a more accurate, less tedious, less time-consuming, and less subjective means than other counting techniques6.
Despite the prominence of the Coulter technique in cell counting, its widespread use in routine biological studies has been prohibitive due to the cost and size of traditional instruments. Although a less expensive Coulter-based instrument has been produced, it has limitations as compared to its more expensive counterparts in the correction for "coincidence events" in which two or more cells pass through the aperture and are measured simultaneously. Another limitation with existing Coulter technologies is the lack of metrics on the overall health of cell samples. Consequently, additional techniques must often be used in conjunction with Coulter counting to assess cell viability. This extends experimental setup time and cost since the traditional methods of viability assessment require cell staining and/or use of expensive and cumbersome equipment such as a flow cytometer.
The Moxi Z mini automated cell counter, described here, is an ultra-small benchtop instrument that combines the accuracy of the Coulter Principle with a thin-film sensor technology to enable precise sizing and counting of particles ranging from 3-25 microns, depending on the cell counting cassette used. The M type cassette can be used to count particles from with average diameters of 4 - 25 microns (dynamic range 2 - 34 microns), and the Type S cassette can be used to count particles with and average diameter of 3 - 20 microns (dynamic range 2 - 26 microns). Since the system uses a volumetric measurement method, the 4-25 microns corresponds to a cell volume range of 34 - 8,180 fL and the 3 - 20 microns corresponds to a cell volume range of 14 - 4200 fL, which is relevant when non-spherical particles are being measured. To perform mammalian cell counts using the Moxi Z, the cells to be counted are first diluted with ORFLO or similar diluent. A cell counting cassette is inserted into the instrument, and the sample is loaded into the port of the cassette. Thousands of cells are pulled, single-file through a "Cell Sensing Zone" (CSZ) in the thin-film membrane over 8-15 seconds. Following the run, the instrument uses proprietary curve-fitting in conjunction with a proprietary software algorithm to provide coincidence event correction along with an assessment of overall culture health by determining the ratio of the number of cells in the population of interest to the total number of particles. The total particle counts include shrunken and broken down dead cells, as well as other debris and contaminants. The results are presented in histogram format with an automatic curve fit, with gates that can be adjusted manually as needed.
Ultimately, the Moxi Z enables counting with a precision and accuracy comparable to a Coulter Z2, the current gold standard, while providing additional culture health information. Furthermore it achieves these results in less time, with a smaller footprint, with significantly easier operation and maintenance, and at a fraction of the cost of comparable technologies.

The Moxi Z miniature automated cell counter is a novel instrument that combines the Coulter Principle with patented thin-film sensor technology and a proprietary software algorithm to perform sizing and counting of a broad size range of particles as well as to determine the overall health of monodisperse mammalian cell cultures. This protocol describes the use of this instrument for counting and assessing the health of cell cultures.

测定哺乳动物细胞计数，细胞的大小和使用末席ž迷你自动细胞计数细胞的健康

Particle and cell counting is used for a variety of applications including routine cell culture, hematological analysis, and industrial controls1-5. A ...

Preparation of Rat Serum Suitable for Mammalian Whole Embryo Culture

Mammalian whole embryo culture (WEC) is a widely used technique for examining pharmacological toxicity in developing mouse and rat embryos and for investigating the mechanisms of developmental processes. Immediately centrifuged (IC) rat serum is commonly used for WEC and is essential for the growth and development of cultured mouse and rat embryos ex vivo. For the culture of midgestation embryos (i.e., E8.0-12.5 for the mouse, and E10.0-14.5 for the rat), 100% rat serum is the best media for supporting the growth of the embryo ex vivo. To prepare rat serum suitable for WEC, the collected blood should be centrifuged immediately to separate the blood cells from the plasma fraction. After centrifugation, the fibrin clot forms in the upper layer; this clot should be squeezed gently using a pair of sterile forceps and subsequently centrifuged to completely separate the blood cells from the serum. In this video article, we demonstrate our standard protocol for the preparation of optimal IC rat serum, including blood collection from the abdominal aorta of male rats and extraction of the serum by centrifugation.

Mammalian whole embryo culture (WEC) is widely used in teratology and developmental biology. Immediately centrifuged rat serum is commonly provided as a medium for both mouse and rat WEC. In this video, we demonstrate our standard protocol for the preparation of high-quality rat serum suitable for mammalian WEC.

大鼠血清适用于哺乳动物全胚胎培养的制备

Mammalian whole embryo culture (WEC) is a widely used technique for examining pharmacological toxicity in developing mouse and rat embryos and for ...

Direct Protein Delivery to Mammalian Cells Using Cell-permeable Cys2-His2 Zinc-finger Domains

Due to their modularity and ability to be reprogrammed to recognize a wide range of DNA sequences, Cys2-His2 zinc-finger DNA-binding domains have emerged as useful tools for targeted genome engineering. Like many other DNA-binding proteins, zinc-fingers also possess the innate ability to cross cell membranes. We recently demonstrated that this intrinsic cell-permeability could be leveraged for intracellular protein delivery. Genetic fusion of zinc-finger motifs leads to efficient transport of protein and enzyme cargo into a broad range of mammalian cell types. Unlike other protein transduction technologies, delivery via zinc-finger domains does not inhibit enzyme activity and leads to high levels of cytosolic delivery. Here a detailed step-by-step protocol is presented for the implementation of zinc-finger technology for protein delivery into mammalian cells. Key steps for achieving high levels of intracellular zinc-finger-mediated delivery are highlighted and strategies for maximizing the performance of this system are discussed.

Direct Protein Delivery to Mammalian Cells Using Cell-permeable Cys2-His2 Zinc-finger Domains

Zinc-finger domains are intrinsically cell-permeable and capable of mediating protein delivery into a broad range of mammalian cell types. Here, a detailed step-by-step protocol for implementing zinc-finger technology for intracellular protein delivery is presented.

蛋白质直接送货到哺乳动物细胞利用细胞渗透半胱氨酸<sub&gt; 2</sub&gt; -His<sub&gt; 2</sub&gt;锌指域

Due to their modularity and ability to be reprogrammed to recognize a wide range of DNA sequences, Cys2-His2 zinc-finger DNA-binding domains have emerged ...