Name: using microfluidic devices to measure lifespan and cellular phenotypes in single budding yeast cells
Uploaded: 2017-03-30T15:00:00.000+00:00
Duration: 9 min 18 s
Description: Watch this Scientific Journal Video about Using Microfluidic Devices to Measure Lifespan and Cellular Phenotypes in Single Budding Yeast Cells at JoVE.com

This research was supported by NIH Grant AG043080 and the National Natural Science Foundation of China (NSFC), No. 11434001. We thank Lucas Waldburger for proofreading the manuscript.

1.硅片模具制造注:光掩膜的设计与AutoCAD软件和商业公司制造的。这种设计包含不同的图案（三层<a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55412/single_cell_trap.dwg" target="_blank">补充文件1</a> ）。第一，第二，和第三层的高度分别为大约4微米，10微米和50微米。硅晶片模具从使用软光刻9,10光掩模创建。 <ol><li>在200℃下烘烤的硅晶片10分钟，以蒸发水蒸汽。旋涂负性光刻胶SU-8到硅晶片上。 注意:旋涂SU-8 3005在5,000rpm下30秒，以产生第一层，SU-8 2010以3,000rpm进行30秒，以产生第二层，和SU-8 3025以2000rpm进行30秒，以产生第三层。 </li><li>软烤吨处理前的涂布晶片他的图案转移。对准，并且使用掩模对准器在暴露"直接接触"模式的晶片。 注:软烘烤的晶片在95℃下2分钟，所述第一层，在95℃下3分钟为第二层，和在95℃下15分钟的第三层。在100mJ / cm 2的第一层，130毫焦耳/厘米2的第二层，和为200mJ / cm 2的第三层使用的曝光剂量。 </li><li>曝光后，后烘烤的晶圆，并使用SU-8开发者开发。干燥使用N 2枪和硬烘烤在200℃下将晶片30分钟的晶片。 注:将硅晶片模具是用透明胶带固定到15厘米直径的塑料培养皿中，与图案的一面朝上。一般，我们把在相同的模具，它允许多个微流体芯片来同时制造多个相同的芯片结构。每个模具可以重新使用多次以制造微流控芯片。 </li></ol> 2.微流控芯片制造<ol><li>放在天平上的清洁称重船和去皮。倾50克PDMS碱入称量皿。 </li><li> 5克PDMS固化剂添加到所述称量皿（w / w至所述PDMS基的1:10的比例）。 注意:此卷是基于一个直径为15cm，培养皿与模具。如果使用不同大小的培养皿中调整试剂的量。 </li><li>搅拌PDMS基并用一次性移液管的固化剂。从称量船的边缘开始慢慢地向内移动。充分搅拌几分钟直至小气泡形成在整个混合物中;彻底混合对于PDMS聚合是必不可少的。 </li><li>把混合物慢慢倒入培养皿;该混合物必须完全覆盖硅晶片模具。 </li><li>放置在真空中的陪替氏培养皿10分钟，以除去从PDMS混合物中的所有气泡。如果气泡保留在混合物的表面上，用移液管吹出来。 </li><li>孵育硅晶片与模具在75℃的烘箱中用于PDMS约2小时。 </li><li>轻轻剥离聚合PDMS断硅晶片模具的层，避免对晶片模具的结构的任何损坏。可替换地，直接地从硅晶片与模具周围使用单边缘工业刀片图案的最小5毫米余量切割PDMS;轻轻剥离PDMS层从晶片模具。 </li><li>放置PDMS层与图案面朝上的替补​​。仔细地切出具有单边缘工业刀片的单独的芯片，保持从每个单芯片，以避免损害结构的边缘足够的裕度。 </li><li>朝上的图案的一面，使用冲头笔（0.75毫米ID），以打孔通过入口和出口圆直倒在通道的每一侧。 注意:这些孔创建介质流动的通路。因此，关键是要经过圈，并通过PDMS层冲一路。确保从孔去除PDMS列。 </li><li>通过再次插入冲笔针头插入孔检查每一个冲孔。确保针头可以从另一面出来，这表明没有堵塞。将胶带粘图案表面，然后轻轻剥离胶带清洁灰尘颗粒。重复此步骤至少三次。留在PDMS一块干净透明胶带，以保持消毒。 </li><li>重复对PDMS的相对侧此过程并离开最后一块胶带上为好。 </li><li>制备一个24毫米×30毫米玻璃盖的厚度为0.13-0.17毫米。喷洒70％的乙醇在玻璃上并干燥与除尘器消毒的表面上;另外，该玻璃可以与高压灭菌水洗涤，并用除尘器干燥。 </li><li>转移盖玻璃和PDMS的塑料板。从PDMS取下胶带并放置图案的一面朝上。避免在传送过程中与图案表面的任何接触。 </li><li&gt;放置在等离子体机的塑料板。应用氧等离子体处理的PDMS和玻璃盖片以使表面亲水性的具有下列操作参数:曝光，75秒;气体稳定，20毫升/分钟;压力，200;和电源，100 W. </li><li>小心地将PDMS到盖玻璃，连接两个亲水表面（即在等离子体处理期间朝上的表面）。确保有是PDMS和玻璃盖之间没有气泡。 </li><li>孵育在烘箱中在PDMS芯片在75℃下至少2小时。 注意:微流体缺陷可能会导致液体渗漏到在实验过程中的显微镜。因此，通过从与镊子的边缘略微抬起PDMS检查PDMS和盖玻璃之间的结合是重要的。轻轻提起PDMS不应它从玻璃盖分开，指示成功的键。 </li></ol> 3.实验准备<ol><l我&gt;管的准备。 <ol><li>淹没在70％乙醇溶液分别PDMS芯片制备前1天的输入和输出管。 </li><li>填充有5毫升的注射器用高压灭菌水清洗管。通过插入注射器上的管，并用水冲洗管洗每个管中。 </li><li>重复该洗涤步骤至少3次以清洁乙醇残留物。 </li></ol></li><li>检查用10X物镜在光学显微镜下的PDMS通道，以确保该结构是一致的和完整的。稳定PDMS芯片上使用透明胶带的显微镜平台。 注:在步骤2中的时间使多个芯片，以确保有备份芯片，特别是如果使设备首次。 </li><li>填充有5毫升的注射器用高压灭菌水。固定注射器的注射泵，并插入相应的输入和输出管。设置洗涤速度至750μL/ h至冲洗芯片约10分钟。在分支通道中的气泡应该在此期间被洗掉;如果有剩余的气泡，手动调整速度以消除气泡。 </li><li>酵母样品制备。 <ol><li>传输1制备的酵母样品（OD 600 0.6-0.8）中的溶液到两个1.5毫升微量离心管中离心5分钟，在3000×g下。 </li><li>除去大部分从每管中的上清液并结合其余以形成0.5毫升的样品。 </li></ol></li><li>暂停注射泵和从PDMS芯片删除输入管。 </li><li>手动反向注射泵吸小气泡（1至2cm长）到每个输入管。移动对酵母​​样品中吸;气泡将显示样品边界，并指出有多少样本已经绘就。 注意:避免将试料吸入到注射器中。 </li><li>插入输入管放回PDMS芯片，并重新启动泵以750μL/ h的速度加载细胞。 </li><li>离开注射器泵上10分钟的n和检查显微镜下细胞加载进展;一个成功加载将捕获细胞下暂停支柱的60％以上。 </li><li>切换到营养溶液和调整速度以用于细胞培养400微升/小时。 </li><li>在显微镜观察选择位置。 注:对于寿命测量，图像通过光学显微镜拍摄一次，每15分钟用40×物镜。对于荧光报告分析，图像通过用60X油浸物镜的荧光显微镜拍摄每15分钟一次。 </li></ol>

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The authors declare that they have no competing financial interests.

PDMS的设备需要新鲜的。否则，引起的插入管插入装置的空气气泡将难以去除。步骤3.4是重要通过浓缩细胞提高电池装载效率。为了增加实验的吞吐量，连接到同一个PDMS芯片上4至6个模块来独立地操作泵通常用于执行4至6个不同的实验（不同菌株或介质的组合物）同时进行。 相比于常规的酵母复制型老化试验（使用显微解剖），这里提出的微流体方法不太费力和耗时的。此外，微流体装置允许各种细胞或分子参数，包括细胞大小，细胞周期动力学，细胞形态，以及各种分子标记的详细的量化。该微流控方法实现长期的细胞通过保留高分辨率显微镜追踪下PDMS微焊盘作为子细胞母细胞被自动冲洗。 该装置使用软PDMS微垫捕获母细胞4，用的基本结构相似，如Huberts 等。在他的作品7中。有许多在该装置的设计和实验方案的差异。在该装置中更广泛的PDMS垫允许被捕获并分析（ 图2和3， 电影1）一些新生子细胞。为了识别这些细胞，我们从注释已经被困母细胞芽子细胞，忽略冲走没有萌芽的子代细胞。平均而言，我们得到了每PDMS垫约2新鲜母细胞。这些新鲜母细胞未知历史的细胞的比例为约1:2，其中约三分之一保持在该装置为全寿命。这些细胞允许更准确的寿命测量中的d使得可以分析母女细胞之间的相关性。在该装置中，更深的主通道连接到在观测由两种较浅侧通道;这样的设计有助于降低被拦截的大气泡的一侧通道的机会。用于微流体芯片的生产，该协议也使用简单的方法来粘结PDMS和玻璃盖片，只是利用等离子体的曝光和烘烤炉，这增加了成功率。 与此协议中，微流体装置能够鲁棒地捕集每PDMS垫的至少一个小区（3-5细胞平均）在实验开始时为野生型单倍体酵母菌株（ 即，BY4741或BY4742）。在细胞的约30％，可在其整个生命周期全被保留。值得注意的是，PDMS装置的性能取决于悬挂柱和玻璃和酵母细胞的大小之间的间隙尺寸。对于野生型单倍体酵母菌株，所述对于该间隙合适的尺寸是3.5-4.5微米。在这个范围之外时，装载效率和细胞保留率大幅下降。因此，对于具有更大或更小的小区尺寸7的酵母菌株新设备必须通过修改在步骤1中所做的第一层的高度来制造。 总之，本文中所描述的设备和协议不仅适用于酵母的老化研究，但也适用于需要母细胞的跟踪和分子标志物的许多代或整个寿命监视其它实验。

芽殖酵母是衰老研究了强大的模式生物。然而，在酵母的常规寿命测定依赖于显微切割，这不仅是劳动密集，而且低吞吐量1,2。此外，他们的年龄在传统的显微方法不提供在单一母细胞的各种细胞和分子特征的详细视图。微流体装置的发展已使自动程序以测量酵母寿命以及跟随的分子标记和各种细胞表型在整个母细胞3，4，5，6，7，8的寿命。后的酵母细胞被加载到微流控装置，它们可以在显微镜下使用自动时间圈跟踪È成像。随着成像处理工具的帮助下，各种细胞和分子的表型可以被提取3，6，8，包括寿命，大小，荧光报道，细胞形态，细胞周期动力学， 等 ，其中许多是难以或不可能获得使用传统的显微解剖法。因为他们的成功发展了几年前3，4，6，7的微流体装置已经获得了在酵母衰老研究突出。一些研究小组已经随后发表在早期的设计5的变化，许多酵母实验室已经采用微流体装置作为研究对象。 在细胞培养进行指数增长，可用于观测岁母细胞的数量是miniscu乐。因此，微流体装置的寿命测量一般设计原则是保留母细胞，并删除子细胞。一个这样的设计使得使用酵母进行不对称细胞分裂的事实。在该装置将捕获更大母细胞的结构和更小的允许子细胞被冲走。这篇文章中描述的微流体芯片采用了软聚二甲基硅氧烷（PDMS）垫（垂直悬挂列）以捕获母细胞（ 图1）。类似的设计的设备先前已被3,4，6,7的报道。该协议使用一个简单的程序来制造微流体装置和被用于延时成像实验优化的直接的细胞装载方法。一个在微流体装置的关键参数是用于捕获母细胞的PDMS焊盘的宽度。我们ðevice使用更宽垫，可以让更多的母细胞各垫下，包括可以在整个他们的寿命进行跟踪新鲜母细胞的显著部分。除了寿命测量，当细胞需要跟踪许多代或当在整个寿命的观察是必要这个协议是用于单细胞延时成像实验是有用的。

<table><tbody><tr><td>3&#039;&#039; &amp;lt;111&gt; silicon wafer</td><td>Addison Engineering</td><td></td><td></td></tr><tr><td>SU-8 2000 and 3000 Series</td><td>MicroChem</td><td></td><td></td></tr><tr><td>Sylgard&amp;reg; 184 Silicone Elastomer Kit</td><td>ellsworth</td><td>2065622</td><td>Include Sylgard&amp;reg; silicone elastomer base and curing agent</td></tr><tr><td>Petri dishes</td><td>VWR</td><td>391-1502</td><td></td></tr><tr><td>Harris Uni-core&amp;trade; punch (I.D. 0.75 mm)</td><td>Sigma-Aldrich</td><td>29002513</td><td></td></tr><tr><td>24 mm x 40 mm SLIP-RITE&amp;reg; cover glass</td><td>Thermo Fisher Scientific</td><td>102440</td><td></td></tr><tr><td>3M Scotch Tape</td><td>ULINE</td><td>S-10223</td><td></td></tr><tr><td>VWR&amp;reg; Razor Blades</td><td>VWR</td><td>55411-050</td><td></td></tr><tr><td>Pure Ethanol, Koptec</td><td>VWR</td><td>64-17-5</td><td></td></tr><tr><td>Whoosh-Duster&amp;trade;</td><td>VWR</td><td>16650-027</td><td></td></tr><tr><td>5 mL BD Syringe (Luer-Lock&amp;trade; Tip)</td><td>Becton, Dickinson and Company</td><td>309646</td><td></td></tr><tr><td>PTFE Standard Wall Tubing (100 ft, AWG Size: 22, Nominal ID: 0.028)</td><td>Component Supply Company</td><td>SWTT-22</td><td></td></tr><tr><td>Needle Assortment</td><td>Component Supply Company</td><td>NEKIT-1</td><td></td></tr><tr><td>Desiccator</td><td>HACH</td><td>2238300</td><td></td></tr><tr><td>Lab Oven</td><td>Fisher Scientific</td><td>13246516GAQ</td><td></td></tr><tr><td>Nikon TE2000 microscope with 40X and 60X objective</td><td>Nikon</td><td></td><td></td></tr><tr><td>Zeiss Axio Observer Z1 with 40X and 60X objective</td><td>Zeiss</td><td></td><td></td></tr><tr><td>Syringe Pump</td><td>Longerpump</td><td>TS-1B</td><td></td></tr></tbody></table>

using microfluidic devices to measure lifespan and cellular phenotypes in single budding yeast cells

Budding yeast Saccharomyces cerevisiae is an important model organism in aging research. Genetic studies have revealed many genes with conserved effects on the lifespan across species. However, the molecular causes of aging and death remain elusive. To gain a systematic understanding of the molecular mechanisms underlying yeast aging, we need high-throughput methods to measure lifespan and to quantify various cellular and molecular phenotypes in single cells. Previously, we developed microfluidic devices to track budding yeast mother cells throughout their lifespan while flushing away newborn daughter cells. This article presents a method for preparing microfluidic chips and for setting up microfluidic experiments. Multiple channels can be used to simultaneously track cells under different conditions or from different yeast strains. A typical setup can track hundreds of cells per channel and allow for high-resolution microscope imaging throughout the lifespan of the cells. Our method also allows detailed characterization of the lifespan, molecular markers, cell morphology, and the cell cycle dynamics of single cells. In addition, our microfluidic device is able to trap a significant amount of fresh mother cells that can be identified by downstream image analysis, making it possible to measure the lifespan with higher accuracy.

实验后，将细胞和许多细胞和分子表型的寿命可以从所记录的时间推移的图像被提取。由于有许多可从每个细胞中提取不同的特征，分析的第一步是将注释细胞和事件，包括位置和小区的边界和正被跟踪的各种事件的定时，例如作为萌芽事件。这些注释将使它更容易返回到相同的一组细胞并分析不同的特点，在未来。使用图像分析软件，如ImageJ的11和相关联的插件，表型的列表然后可以从使用的小区的记录注解的图像数据中提取。 以下是与微流体装置测量的表型的几个例子。酵母的寿命表型可通过计算由每个捕获母细胞中产生芽的数量和与所述的Kaplan-Meier估计器估计来获得。最初被困在微流体装置的细胞是未知的青睐。为了最小化从这些未知年龄始发寿命测量值的偏差，以前的方法使用在捕获的细胞芽疤痕的平均数目来校准寿命4,7。然而，芽痕测量需要细胞的额外染色，并提供了偏置的间接估计。使用该协议，设备经常陷阱从已捕获的细胞芽关闭新鲜的子细胞。然后，这些细胞变成母细胞。这种细胞可以使用我们的下游图像分析软件（ 电影1）来识别。这些新鲜母细胞提供更精确的测量寿命。 如图2所示，新鲜母细胞的寿命曲线是COMPA红色与年龄不详的细胞。在这个实验中，新鲜母细胞的平均寿命稍长（约2代差的中位寿命）。 除了芽，其他有趣的表型，如在两个连续事件出芽之间的时间间隔，如在图3中示出的数目，可以从成像数据3，7，8中提取。单元格中输入以下几个较慢的初始芽接一段较快的萌芽。含苞待放的放缓极大地朝其寿命结束，表明这些老化细胞的非正常状态。细胞周期动力学包含有关老少细胞的细胞状态非常有用的信息，并已使用，例如，表征端粒酶突变体12。重要的是，该设备可以是用于测量荧光信号在整个寿命（ 图4），允许的分子标记，其可以是老化过程的驱动器的跟踪。 <img alt="图1" src="/files/ftp_upload/55412/55412fig1.jpg" /> 图1: 在微流体装置的设计的示意图。该装置由可以并行操作6个独立的功能模块。每个模块由连接到两个侧信道的一个主通道。每个侧通道具有113个悬挂列。一个额外的桥在中间加入到连接与两个侧通道的主通道。母细胞被捕获在悬挂柱的下方，而较小的子细胞被冲走流。 <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55412/55412fig1large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> 图2: 新鲜母细胞的寿命比未知的历史较长的细胞。新鲜母细胞与未知的历史细胞的复制寿命（电池被困在实验开始时）在SD培养基中，30度。平均而言，新鲜母细胞活约2代更长的时间。 <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55412/55412fig2large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图3" src="/files/ftp_upload/55412/55412fig3.jpg" /> 图3: 出芽间隔变化老化电池的长度。作为芽接之间的时间帧测量出芽的间隔颜色编码，将细胞通过它们的复制寿命排序，并且新鲜母细胞九月来自未知历史的细胞相分离。 <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55412/55412fig3large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图4" src="/files/ftp_upload/55412/55412fig4.jpg" /> 图4: 使用微流体装置测量热激因子1（HSF1）的活性。的HSF1活性报告基因是通过融合到一个残缺CYC1启动子与HSE上游13绿色荧光蛋白（GFP）构成。荧光图像每隔30分钟。 GFP强度然后使用定制的MATLAB代码14定量。对于每个单细胞数据点连接起来，并用彩色线表示。在该实验中，将菌株在30℃下生长在SD培养基中含有2％葡萄糖（重量/体积）过夜;然后将其用为恢复相同的培养基中稀释。之后，用0.05％葡萄糖（重量/体积）的SD培养基中使用的荧光测量期间生长细胞在微流体芯片中。 <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55412/55412fig4large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图4" src="/files/ftp_upload/55412/55412mov1.jpg" /> 电影1: 新鲜母细胞的一个例子被截留在装置内对整个寿命。影片显示了一个被困的细胞诞生一个新的母细胞。在电影开头的红色箭头标志的新鲜母细胞和它的第一个萌芽的位置。该母细胞被困在其整个生命周期的微流体装置。 <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55412/55412movie1.mov" target="_blank">请点击此处观看该视频。 （右键点击下载）。</a>

Watch this Scientific Journal Video about Using Microfluidic Devices to Measure Lifespan and Cellular Phenotypes in Single Budding Yeast Cells at JoVE.com

Using Microfluidic Devices to Measure Lifespan and Cellular Phenotypes in Single Budding Yeast Cells

本文介绍用于生产微流体芯片和微流体实验的设置来测量的寿命和单个酵母细胞的细胞表型进行了优化的协议。

使用微流体装置来衡量寿命和细胞表型单芽殖酵母细胞

Budding yeast Saccharomyces cerevisiae is an important model organism in aging research. Genetic studies have revealed many genes with conserved effects ...

using-microfluidic-devices-to-measure-lifespan-cellular-phenotypes

Nanyang Technological University

Research

JoVE Journal

Biology

7.7K 次观看.  Peking University. 本文介绍用于生产微流体芯片和微流体实验的设置来测量的寿命和单个酵母细胞的细胞表型进行了优化的协议。 

视频: Using Microfluidic Devices to Measure Lifespan and Cellular Phenotypes in Single Budding Yeast Cells

A Microfluidic Device for Studying Multiple Distinct Strains

The study of cell responses to environmental changes poses many experimental challenges: cells need to be imaged under changing conditions, often in a comparative manner. Multiwell plates are routinely used to compare many different strains or cell lines, but allow limited control over the environment dynamics. Microfluidic devices, on the other hand, allow exquisite dynamic control over the surrounding conditions, but it is challenging to image and distinguish more than a few strains in them. Here we describe a method to easily and rapidly manufacture a microfluidic device capable of applying dynamically changing conditions to multiple distinct yeast strains in one channel. The device is designed and manufactured by simple means without the need for soft lithography. It is composed of a Y-shaped flow channel attached to a second layer harboring microwells. The strains are placed in separate microwells, and imaged under the exact same dynamic conditions. We demonstrate the use of the device for measuring protein localization responses to pulses of nutrient changes in different yeast strains.

We present a simple method to produce microfluidic devices capable of applying similar dynamic conditions to multiple distinct strains, without the need for a clean room or soft lithography.

一种微流体装置研究多个不同的菌株

The study of cell responses to environmental changes poses many experimental challenges: cells need to be imaged under changing conditions, often in a ...

科研

生物工程

Continuous High-resolution Microscopic Observation of Replicative Aging in Budding Yeast

We demonstrate the use of a simple microfluidic setup, in which single budding yeast cells can be tracked throughout their entire lifespan. The microfluidic chip exploits the size difference between mother and daughter cells using an array of micropads. Upon loading, cells are trapped underneath these micropads, because the distance between the micropad and cover glass is similar to the diameter of a yeast cell (3-4 &mu;m). After the loading procedure, culture medium is continuously flushed through the chip, which not only creates a constant and defined environment throughout the entire experiment, but also flushes out the emerging daughter cells, which are not retained underneath the pads due to their smaller size. The setup retains mother cells so efficiently that in a single experiment up to 50 individual cells can be monitored in a fully automated manner for 5 days or, if necessary, longer. In addition, the excellent optical properties of the chip allow high-resolution imaging of cells during the entire aging process.

We describe here the operation of a microfluidic device that allows continuous and high-resolution microscopic imaging of single budding yeast cells during their complete replicative and/or chronological lifespan.

在芽殖酵母连续高分辨率显微观察复制性衰老

We demonstrate the use of a simple microfluidic setup, in which single budding yeast cells can be tracked throughout their entire lifespan. The ...

Microfluidic Picoliter Bioreactor for Microbial Single-cell Analysis: Fabrication, System Setup, and Operation

In this protocol the fabrication, experimental setup and basic operation of the recently introduced microfluidic picoliter bioreactor (PLBR) is described in detail. The PLBR can be utilized for the analysis of single bacteria and microcolonies to investigate biotechnological and microbiological related questions concerning, e.g.&#160;cell growth, morphology, stress response, and metabolite or protein production on single-cell level. The device features continuous media flow enabling constant environmental conditions for perturbation studies, but in addition allows fast medium changes as well as oscillating conditions to mimic any desired environmental situation. To fabricate the single use devices, a silicon wafer containing sub micrometer sized SU-8 structures served as the replication mold for rapid polydimethylsiloxane casting. Chips were cut, assembled, connected, and set up onto a high resolution and fully automated microscope suited for time-lapse imaging, a powerful tool for spatio-temporal cell analysis. Here, the biotechnological platform organism Corynebacterium glutamicum&#160;was seeded into the PLBR and cell growth and intracellular fluorescence were followed over several hours unraveling time dependent population heterogeneity on single-cell level, not possible with conventional analysis methods such as flow cytometry. Besides insights into device fabrication, furthermore, the preparation of the preculture, loading, trapping of bacteria, and the PLBR cultivation of single cells and colonies is demonstrated. These devices will add a new dimension in microbiological research to analyze time dependent phenomena of single bacteria under tight environmental control. Due to the simple and relatively short fabrication process the technology can be easily adapted at any microfluidics lab and simply tailored towards specific needs.

In this protocol the fabrication, setup and basic operation of a microfluidic picoliter bioreactor (PLBR) for single-cell analysis of prokaryotic microorganisms is introduced. Industrially relevant microorganisms were analyzed as proof of principle allowing insights into growth rate, morphology, and phenotypic heterogeneity over certain time periods, hardly possible with conventional methods.

In this protocol the fabrication, experimental setup and basic operation of the recently introduced microfluidic picoliter bioreactor (PLBR) is described ...

Quantifying Yeast Chronological Life Span by Outgrowth of Aged Cells

The budding yeast Saccharomyces cerevisiae has proven to be an important model organism in the field of aging research 1. The replicative and chronological life spans are two established paradigms used to study aging in yeast. Replicative aging is defined as the number of daughter cells a single yeast mother cell produces before senescence; chronological aging is defined by the length of time cells can survive in a non-dividing, quiescence-like state 2. We have developed a high-throughput method for quantitative measurement of chronological life span. This method involves aging the cells in a defined medium under agitation and at constant temperature. At each age-point, a sub-population of cells is removed from the aging culture and inoculated into rich growth medium. A high-resolution growth curve is then obtained for this sub-population of aged cells using a Bioscreen C MBR machine. An algorithm is then applied to determine the relative proportion of viable cells in each sub-population based on the growth kinetics at each age-point. This method requires substantially less time and resources compared to other chronological lifespan assays while maintaining reproducibility and precision. The high-throughput nature of this assay should allow for large-scale genetic and chemical screens to identify novel longevity modifiers for further testing in more complex organisms.

Chronological aging in yeast refers to the loss of cell viability associated with time in stationary phase. Here we describe a high-throughput method for quantitatively determining yeast chronological life span.

量化老化细胞生长的酵母年谱跨度

The budding yeast Saccharomyces cerevisiae has proven to be an important model organism in the field of aging research 1. The replicative and ...

生物学

Measuring Replicative Life Span in the Budding Yeast

Aging is a degenerative process characterized by a progressive deterioration of cellular components and organelles resulting in mortality. The budding yeast Saccharomyces cerevisiae has been used extensively to study the biology of aging, and several determinants of yeast longevity have been shown to be conserved in multicellular eukaryotes, including worms, flies, and mice 1. Due to the lack of easily quantified age-associated phenotypes, aging in yeast has been assayed almost exclusively by measuring the life span of cells in different contexts, with two different life span paradigms in common usage 2. Chronological life span refers to the length of time that a mother cell can survive in a non-dividing, quiescence-like state, and is proposed to serve as a model for aging of post-mitotic cells in multicellular eukaryotes. Replicative life span, in contrast, refers the number of daughter cells produced by a mother cell prior to senescence, and is thought to provide a model of aging in mitotically active cells. Here we present a generalized protocol for measuring the replicative life span of budding yeast mother cells. The goal of the replicative life span assay is to determine how many times each mother cell buds. The mother and daughter cells can be easily differentiated by an experienced researcher using a standard light microscope (total magnification 160X), such as the Zeiss Axioscope 40 or another comparable model. Physical separation of daughter cells from mother cells is achieved using a manual micromanipulator equipped with a fiber-optic needle. Typical laboratory yeast strains produce 20-30 daughter cells per mother and one life span experiment requires 2-3 weeks.

In this article we present a general protocol for measuring the replicative life span of yeast mother cells.

在芽殖酵母中的自我复制寿命测量

Aging is a degenerative process characterized by a progressive deterioration of cellular components and organelles resulting in mortality. The budding ...

A Suppressor Screen for the Characterization of Genetic Links Regulating Chronological Lifespan in Saccharomyces cerevisiae

Aging is the time dependent deterioration of an organism&#8217;s normal biological processes that increases the probability of death. Many genetic factors contribute to alterations in the normal aging process. These factors intersect in complex ways, as evidenced by the wealth of documented links identified and conserved in many organisms. Most of these studies focus on loss-of-function, null mutants that allow for rapid screening of many genes simultaneously. There is much less work that focuses on characterizing the role that overexpression of a gene in this process. In the present work, we present a straightforward methodology to identify and clone genes in the budding yeast, Saccharomyces cerevisiae, for study in suppression of the short-lived chronological lifespan phenotype seen in many genetic backgrounds. This protocol is designed to be accessible to researchers from a wide variety of backgrounds and at various academic stages. The SIR2 gene, which codes for a histone deacetylase, was selected for cloning in the pRS315 vector, as there have been conflicting reports on its effect on the chronological lifespan. SIR2 also plays a role in autophagy, which results when disrupted via the deletion of several genes, including the transcription factor ATG1. As a proof of principle, we clone the SIR2 gene to perform a suppressor screen on the shortened lifespan phenotype characteristic of the autophagy deficient atg1&#916; mutant and compare it to an otherwise isogenic, wild type genetic background.

A Suppressor Screen for the Characterization of Genetic Links Regulating Chronological Lifespan in Saccharomyces cerevisiae

Here is a protocol to identify genetic interactions through an increased copy number suppressor screen in Saccharomyces cerevisiae. This method allows researchers to identify, clone, and test suppressors in short-lived yeast mutants. We test the effect of the copy number increase of SIR2 on lifespan in an autophagy null mutant.

糖精中遗传链接调控时间寿命的抑制 器屏幕

Aging is the time dependent deterioration of an organism&#8217;s normal biological processes that increases the probability of death. Many genetic factors ...

遗传学

Studying Age-dependent Genomic Instability using the S. cerevisiae Chronological Lifespan Model

Studies using the Saccharomyces cerevisiae aging model have uncovered life span regulatory pathways that are partially conserved in higher eukaryotes1-2. The simplicity and power of the yeast aging model can also be explored to study DNA damage and genome maintenance as well as their contributions to diseases during aging. Here, we describe a system to study age-dependent DNA mutations, including base substitutions, frame-shift mutations, gross chromosomal rearrangements, and homologous/homeologous recombination, as well as nuclear DNA repair activity by combining the yeast chronological life span with simple DNA damage and mutation assays. The methods described here should facilitate the identification of genes/pathways that regulate genomic instability and the mechanisms that underlie age-dependent DNA mutations and cancer in mammals.

Studying Age-dependent Genomic Instability using the S. cerevisiae Chronological Lifespan Model

Here we describe a set of DNA mutation assays that can be combined with the yeast chronological life span model to study the genes/pathways that regulate or contribute to genomic DNA instability during aging.

研究年龄相关的基因组不稳定性使用 S。酵母年代的寿命模型

Studies using the Saccharomyces cerevisiae aging model have uncovered life span regulatory pathways that are partially conserved in higher eukaryotes1-2. ...

Acquiring Fluorescence Time-lapse Movies of Budding Yeast and Analyzing Single-cell Dynamics using GRAFTS

Fluorescence time-lapse microscopy has become a powerful tool in the study of many biological processes at the single-cell level. In particular, movies depicting the temporal dependence of gene expression provide insight into the dynamics of its regulation; however, there are many technical challenges to obtaining and analyzing fluorescence movies of single cells. We describe here a simple protocol using a commercially available microfluidic culture device to generate such data, and a MATLAB-based, graphical user interface (GUI) -based software package to quantify the fluorescence images. The software segments and tracks cells, enables the user to visually curate errors in the data, and automatically assigns lineage and division times. The GUI further analyzes the time series to produce whole cell traces as well as their first and second time derivatives. While the software was designed for S. cerevisiae, its modularity and versatility should allow it to serve as a platform for studying other cell types with few modifications.

We present a simple protocol to obtain fluorescence microscopy movies of growing yeast cells, and a GUI-based software package to extract single-cell time series data. The analysis includes automated lineage and division time assignment integrated with visual inspection and manual curation of tracked data.

获取荧光时间推移电影的芽殖酵母和分析单细胞动力学移植物

Fluorescence time-lapse microscopy has become a powerful tool in the study of many biological processes at the single-cell level. In particular, movies ...

ODELAY: A Large-scale Method for Multi-parameter Quantification of Yeast Growth

Growth phenotypes of microorganisms are a strong indicator of their underlying genetic fitness and can be segregated into 3 growth regimes: lag-phase, log-phase, and stationary-phase. Each growth phase can reveal different aspects of fitness that are related to various environmental and genetic conditions. High-resolution and quantitative measurements of all 3 phases of growth are generally difficult to obtain. Here we present a detailed method to characterize all 3 growth phases on solid media using an assay called One-cell Doubling Evaluation of Living Arrays of Yeast (ODELAY). ODELAY quantifies growth phenotypes of individual cells growing into colonies on solid media using time-lapse microscopy. This method can directly observe population heterogeneity with each growth parameter in genetically identical cells growing into colonies. This population heterogeneity offers a unique perspective for understanding genetic and epigenetic regulation, and responses to genetic and environmental perturbations. While the ODELAY method is demonstrated using yeast, it can be utilized on any colony forming microorganism that is visible by bright field microscopy.

We present a method for quantifying growth phenotypes of individual yeast cells as they grow into colonies on solid media using time-lapse microscopy termed, One-cell Doubling Evaluation of Living Arrays of Yeast (ODELAY). Population heterogeneity of genetically identical cells growing into colonies can be directly observed and quantified.

ODELAY:用于酵母生长多参数定量的大规模方法

Growth phenotypes of microorganisms are a strong indicator of their underlying genetic fitness and can be segregated into 3 growth regimes: lag-phase, ...

Single-cell Microfluidic Analysis of Bacillus subtilis

Microfluidic technology overcomes many of the limitations to traditional analytical methods in microbiology. Unlike bulk-culture methods, it offers single-cell resolution and long observation times spanning hundreds of generations; unlike agarose pad-based microscopy, it has uniform growth conditions that can be tightly controlled. Because the continuous flow of growth medium isolates the cells in a microfluidic device from unpredictable variations in the local chemical environment caused by cell growth and metabolism, authentic changes in gene expression and cell growth in response to specific stimuli can be more confidently observed. Bacillus subtilis is used here as a model bacterial species to demonstrate a &#34;mother machine&#34;-type method for cellular analysis. We show how to construct and plumb a microfluidic device, load it with cells, initiate microscopic imaging, and expose cells to a stimulus by switching from one growth medium to another. A stress-responsive reporter is used as an example to reveal the type of data that may be obtained by this method. We also briefly discuss further applications of this method for other types of experiments, such as analysis of bacterial sporulation.

Single-cell Microfluidic Analysis of Bacillus subtilis

We present a method for the microfluidic analysis of individual bacterial cell lineages using Bacillus subtilis as an example. The method overcomes shortcomings of traditional analytical methods in microbiology by allowing observation of hundreds of cell generations under tightly controllable and uniform growth conditions.

单细胞微流控芯片分析枯草芽孢杆菌

Microfluidic technology overcomes many of the limitations to traditional analytical methods in microbiology. Unlike bulk-culture methods, it offers ...

使用微流体装置来衡量寿命和细胞表型单芽殖酵母细胞

摘要

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此视频中的章节

一种微流体装置研究多个不同的菌株

在芽殖酵母连续高分辨率显微观察复制性衰老

Microfluidic Picoliter Bioreactor for Microbial Single-cell Analysis: Fabrication, System Setup, and Operation

量化老化细胞生长的酵母年谱跨度

在芽殖酵母中的自我复制寿命测量

糖精中遗传链接调控时间寿命的抑制器屏幕

研究年龄相关的基因组不稳定性使用 S。酵母年代的寿命模型

获取荧光时间推移电影的芽殖酵母和分析单细胞动力学移植物

ODELAY:用于酵母生长多参数定量的大规模方法

单细胞微流控芯片分析枯草芽孢杆菌

使用微流体装置来衡量寿命和细胞表型单芽殖酵母细胞

摘要

探索更多视频

此视频中的章节

一种微流体装置研究多个不同的菌株

在芽殖酵母连续高分辨率显微观察复制性衰老

Microfluidic Picoliter Bioreactor for Microbial Single-cell Analysis: Fabrication, System Setup, and Operation

量化老化细胞生长的酵母年谱跨度

在芽殖酵母中的自我复制寿命测量

糖精中遗传链接调控时间寿命的抑制 器屏幕

研究年龄相关的基因组不稳定性使用 S。酵母年代的寿命模型

获取荧光时间推移电影的芽殖酵母和分析单细胞动力学移植物

ODELAY:用于酵母生长多参数定量的大规模方法

单细胞微流控芯片分析枯草芽孢杆菌

糖精中遗传链接调控时间寿命的抑制器屏幕