使用微流体装置来衡量寿命和细胞表型单芽殖酵母细胞
Summary
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。
Abstract
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.
Introduction
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。
Protocol
Representative Results
Discussion
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?…
Divulgazioni
The authors have nothing to disclose.
Acknowledgements
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.
Materials
| 3'' <111> silicon wafer | Addison Engineering | ||
| SU-8 2000 and 3000 Series | MicroChem | ||
| SYLGARD® 184 SILICONE ELASTOMER KIT | ellsworth | 2065622 | Include Sylgard® silicone elastomer base and curing agent |
| Petri dishes | VWR | 391-1502 | |
| Harris Uni-core™ punch(I.D. 0.75 mm) | Sigma-Aldrich | 29002513 | |
| 24 mm x 40 mm SLIP-RITE® cover glass | Thermo Fisher Scientific | 102440 | |
| 3M Scotch Tape | ULINE | S-10223 | |
| VWR® Razor Blades | VWR | 55411-050 | |
| PURE ETHANOL, KOPTEC | VWR | 64-17-5 | |
| WHOOSH-DUSTER™ | VWR | 16650-027 | |
| 5mL BD Syringe (Luer-Lock™Tip) | Becton, Dickinson and Company. | 309646 | |
| PTFE Standard Wall Tubing (100ft, AWG Size:22, Nominal ID: 0.028) | COMPONENT SUPPLY COMPANY | SWTT-22 | |
| Needle Assortment | COMPONENT SUPPLY COMPANY | NEKIT-1 | |
| Desiccator | HACH | 2238300 | |
| Lab Oven | FISHER SCIENTIFIC | 13246516GAQ | |
| Nikon TE2000 microscope with 40x and 60x objective | Nikon | ||
| Zeiss Axio Observer Z1 with 40x and 60x objective | Zeiss | ||
| Syringe Pump | Longerpump | TS-1B |
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