这项工作得到了美国国立卫生研究院资助R35GM128873和美国国家科学基金会资助2045493（授予MNM）的支持。梅根·妮可·麦克莱恩（Megan Nicole McClean）博士在巴勒斯惠康基金会（Burroughs Wellcome Fund）的科学界面（Scientific Interface）上获得了职业奖。Z.P.H. 得到了 NHGRI 对基因组科学培训计划 5T32HG002760 的培训资助。我们感谢与麦克莱恩实验室成员进行的富有成效的讨论，特别是感谢基兰·斯威尼（Kieran Sweeney）对手稿的评论。

本研究中使用的酵母菌株记录在 材料表中。这些菌株在 22 °C 至 30 °C 的温度范围内表现出强劲的生长，并且可以在各种标准酵母培养基中培养。
1. 设置自动化工作站
<ol><li>为自动化工作站配备能够移动微孔板的机器人夹持臂（RGA，参见 材料表）（图 1）。</li>
	<li>将微孔板加热器振荡器（见材料表）安装到自动化工作站（图1（1））中，并带有提供RGA访问的自动板锁定机构。</li>
	<li>将酶标仪放置在自动化工作站附近（图 1（2）），确保 RGA 可访问性。</li>
	<li>结合微孔板照明装置（图1（3）），方便RGA访问。示例包括 optoPlate8 （如本研究中使用的）或 LITOS9 （参见 材料表）。</li>
</ol>2、照明装置的准备
<ol><li>按照既定方法8、10、11 构建和校准 optoPlate（或替代照明设备）。</li>
	<li>利用照明设备上的适配器启用 RGA 访问。</li>
	<li>使用电子表格输入10 或图形界面12 对 optoPlate 进行编程。按照步骤 3 执行光刺激程序。</li>
</ol>3. 设计光刺激程序
<ol><li>确定实验所需的光照条件。</li>
	<li>在电子表格中输入所需的光照条件，包括光强度、光开始时间、脉冲长度、脉冲数和脉冲间持续时间。 注意:使用 GitHub 存储库中提供的说明将此信息刷入 optoPlate:github.com/mccleanlab/Optoplate-96。请记住，样品板在酶标仪或加热器振荡器上不会被照亮。这些事件的持续时间和频率可能需要根据特定的实验要求进行优化。</li>
	<li>包括每个菌株的黑暗条件，以便进行适当的背景测量。</li>
	<li>对于评估转化体功能的初始表征实验，请使用高光强度。 注意:应针对更敏感的实验优化光强度，因为过多的光可能对酵母13 产生光毒性。</li>
</ol>4.酶标仪的准备
<ol><li>在进行实验之前，配置酶标仪以测量所需的目标量。 注:在本例中，酶标仪被配置为测量由目标菌株表达的报告基因的荧光。根据实验要求，可以使用其他输出，例如发光或光密度。</li>
	<li>在合成完全（SC）培养基14 （或另一种低荧光培养基）（参见 材料表）中培养目标菌株（连同非荧光对照）至要测量的最高细胞密度。将培养物转移到玻璃底黑壁微孔板中（参见 材料表）并测量它们以确定最佳的酶标仪设置。 注意: 通过正确输入板尺寸并从下方测量板来确保读数准确。</li>
	<li>参考荧光蛋白数据库（例如，fpbase.org）以获得目标荧光蛋白15的近似吸收和发射光谱。</li>
	<li>通过对含有荧光和非荧光菌株的孔进行z扫描来确定z值（板和读数器之间的距离）。选择产生最高信噪比的 z 值。</li>
	<li>通过对荧光和非荧光菌株进行吸收和发射扫描来优化吸收和发射光谱，以确定最佳信噪比。</li>
	<li>在增益设置为最佳水平的情况下测量荧光应变，确定可以使用的最高光学增益，而不会导致溢出测量误差。 注意:在涉及相同应变的实验中，应手动一致地设置此光学增益，以确保结果的一致性。</li>
	<li>在酶标仪软件中准备测量脚本（参见 图2）。该脚本将仪器配置为测量培养物的光密度和任何待测荧光蛋白的荧光光谱。 注:在600nm处测量菌株的光密度，除非它们表达红色荧光蛋白。对于表达红色荧光蛋白的菌株，测量 700 nm 处的光密度以避免偏差16。</li>
	<li>设置测量脚本以在测量过程中保持 30 °C 的内部孵育温度。</li>
	<li>根据偏好配置测量脚本以将数据导出到电子表格或 ASCII 文件中。</li>
</ol>5. 机器人编程
<ol><li>根据载体（例如，加热器振荡器、巢平台、照明设备）和实验室器皿（即 96 孔板）的物理布局，在自动化工作站软件中配置工作台定义（参见 材料表）。按照以下步骤在自动化工作站软件中创建一个脚本来执行光感应和测量（图 3）。</li>
	<li>禁用任何内部照明源，以防止光遗传学系统的背景激活。</li>
	<li>将加热器摇床设置为保持 30 °C 的温度。 当板不在加热器振动器上时，它将处于环境温度 （22 °C）。</li>
	<li>利用循环、计时器和循环计数变量定期重复诱导和测量细胞的步骤。</li>
	<li>在记录测量值之前，摇晃样品板以确保所有细胞的正确悬浮。以 1,000 rpm 的速度以 2 mm 的轨道振荡 60 秒通常足以重悬酿酒 酵母 细胞并避免测量偏差。</li>
	<li>使用机械臂将样品板移动到酶标仪上，并取下盖子（如果适用），以防止光密度测量出现偏差。如果酶标仪托架定义设置为不允许盖子，控制软件将自动将盖子取下并更换到指定位置。</li>
	<li>按照步骤 4 中的说明执行酶标仪测量脚本。</li>
	<li>盖上样品板上的盖子，然后使用机械臂将板移动到照明装置上。</li>
	<li>将脚本设置为等待，直到计时器达到指定的时间间隔（例如，30 分钟乘以循环计数变量），然后重复整个循环以获得所需的迭代次数（例如，48 小时实验为 24 次）。</li>
	<li>排除潜在错误，确保载体和实验室器皿定义的正常运行，以及 RGA 通过多次运行空板循环来准确拾取和放置板的能力。</li>
	<li>配置用户警报，以通知用户在协议执行期间可能发生的任何仪器状态更改或错误。</li>
</ol>6. 设置样品板
<ol><li>在富培养基平板上培养酵母菌株，例如YPD琼脂17 （参见 材料表）。包括非荧光（阴性）对照。</li>
	<li>从平板中选择菌落，并将它们接种到玻璃培养管中的 3 mL SC 培养基14 （或其他低荧光培养基，如 LFM18）中。将培养物在30°C下在滚筒上孵育过夜，同时将它们保持在黑暗或无响应的光条件下（例如，红光用于蓝光响应系统）。</li>
	<li>通过将每种培养物的200μL稀释到1mL的SC培养基中，并使用分光光度计或酶标仪记录600nm处的光密度（OD 600）来测量过夜培养物的光密度。</li>
	<li>在玻璃培养管中将每个过夜培养物稀释至OD600 0.1。如需更高通量的菌株测试，请在微孔板中进行自动稀释。</li>
	<li>将稀释的培养物移入 96 孔板中。为每种条件（即，具有相同应变和光照条件的三个相同孔）包括一式三份孔，以考虑技术差异。包括含有空白培养基和非荧光细胞的孔作为阴性对照，以测量背景荧光和光密度。</li>
	<li>在开始光诱导实验之前，将板在30°C下振荡孵育5小时。根据需要调整稀释量和孵育时间，以针对特定菌株和实验条件进行优化。</li>
</ol>7. 执行实验
<ol><li>将样品板放在加热器振荡器上，并按照步骤 5 中的说明启动自动化脚本。</li>
	<li>记录读板器上的初始测量值后，按照步骤 3 中的说明开始光刺激程序。</li>
	<li>如果自动化工作站不在黑暗的房间内，请用遮光窗帘遮住它，以避免背景照明。</li>
</ol>8. 数据分析
<ol><li>为实验创建电子表格图，反映 96 孔板的 8 x 12 布局。确保地图包括一个网格中要测量的应变的名称以及另一个网格中使用的光照条件的描述。</li>
	<li>利用 Python 脚本或其他首选编程语言来分析导出的数据。将地图电子表格导入到数组中，将菌株名称和条件名称与板的每个孔相关联。 注意:用于分析的示例代码可在以下位置找到:https://github.com/mccleanlab/Lustro。或者，可以使用应用程序来解析来自各种酶标仪19的数据。</li>
	<li>将导出的实验电子表格中的数据读入另一个数组。</li>
	<li>生成每种菌株和条件随时间变化的光密度值和荧光值图， 如图 4 所示。</li>
	<li>检查光密度图以确定培养物中指数增长或饱和的阶段。这些信息将有助于选择适当的时间点来比较菌株或条件之间的荧光测量， 如图5所示。</li>
</ol>

使用 Lustro 自动化技术进行酵母光遗传学反应分析

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作者没有什么可透露的。

本文介绍的 Lustro 协议可自动执行培养、照明和测量过程，从而实现光遗传学系统的高通量筛选和表征6。这是通过将照明设备、酶标仪和摇动设备集成到自动化工作站中来实现的。该协议特别证明了Lustro在筛选整合到 酵母酿酒酵 母中的不同光遗传学结构和比较光诱导程序的效用。
该协议中强调的几个关键步骤对于有效利用 Lustro 至关重要。仔细设计?...

光遗传学是一种强大的技术，它利用光敏蛋白来高精度地控制细胞的行为 1,2,3。然而，光遗传学结构的原型设计和确定最佳照明条件可能非常耗时，因此很难优化光遗传学系统4,5。快速筛选和表征光遗传学系统活性的高通量方法可以加快设计-构建-测试周期，以对结构进行原型设计和探索其功能。
Lustro 平台是一种实验室自动化技术，专为光遗传学系统的高通量筛选和表征而设计。它将酶标仪、照明装置和振荡装置与自动化工作站6集成在一起。Lustro 结合了微孔板中细胞的自动培养和光刺激（图 1 和补充图 1），能够快速筛选和比较不同的光遗传学系统。Lustro 平台具有很强的适应性，可以推广到与其他实验室自动化机器人、照明设备、酶标仪、细胞类型和光遗传学系统（包括对不同波长的光有响应的机器人）配合使用。
该协议演示了Lustro用于表征光遗传学系统的设置和使用。以酵母中分裂转录因子的光遗传学控制为例系统，通过探测光输入与荧光报告基因 mScarlet-I7 表达之间的关系来说明该平台的功能和效用。通过遵循该协议，研究人员可以简化光遗传学系统的优化，并加速发现生物系统动态控制的新策略。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>96-well glass bottom plate with&#160; #1.5 cover glass</td>
			<td>Cellvis</td>
			<td>P96-1.5H-N</td>
			<td></td>
		</tr>
		<tr>
			<td>BioShake 3000-T elm (heater shaker)</td>
			<td>QINSTRUMENTS</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Fluent Automation Workstation</td>
			<td>Tecan</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>LITOS (alternative illumination device)</td>
			<td>Hohener, et al. Scientific Reports. 2022</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>optoPlate-96 (illumination device)</td>
			<td>Bugaj, et al. Nature Protocols. 2019</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Robotic Gripper Arm</td>
			<td>Tecan</td>
			<td></td>
			<td>Standard or long Z axes; regular gripper head or automatic Finger Exchange System gripper head, both with a choice of gripper fingers &#8211; eccentric, long eccentric, centric, tube; barcode reader option</td>
		</tr>
		<tr>
			<td>Spark (plate reader)</td>
			<td>Tecan</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Synthetic Complete media</td>
			<td>SigmaAldrich</td>
			<td>Y1250</td>
			<td></td>
		</tr>
		<tr>
			<td>Tecan Connect (user alert app)</td>
			<td>Tecan</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>yMM1734 (BY4741 Mat&#945; ura3&#916;0::5&#39; Ura3 homology, pRPL18B-Gal4DBD-eMagA-tENO1, pRPL18B-eMagB-Gal4AD-tENO1, pGAL1-mScarlet-I-tENO1, Ura3, Ura 3&#39; homology&#160; his3D1 leu2D0 lys2D0 gal80::KANMX gal4::spHIS5)</td>
			<td>Harmer, et al. ACS Syn Bio. 2023</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>yMM1763 (BY4741 Mat&#945; ura3&#916;0::5&#39; Ura3 homology, pRPL18B-Gal4DBD-CRY2(535)-tENO1, pRPL18B-Gal4AD-CIB1-tENO1, pGAL1-mScarlet-I-tENO1, Ura3, Ura 3&#39; homology&#160; his3D1 leu2D0 lys2D0 gal80::KANMX gal4::spHIS5)</td>
			<td>Harmer, et al. ACS Syn Bio. 2023</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>yMM1765 (BY4741 Mat&#945; ura3&#916;0::5&#39; Ura3 homology, pRPL18B-Gal4DBD-eMagA-tENO1, pRPL18B-eMagBM-Gal4AD-tENO1, pGAL1-mScarlet-I-tENO1, Ura3, Ura 3&#39; homology&#160; his3D1 leu2D0 lys2D0 gal80::KANMX gal4::spHIS5)</td>
			<td>Harmer, et al. ACS Syn Bio. 2023</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>YPD Agar</td>
			<td>SigmaAldrich</td>
			<td>Y1500</td>
			<td></td>
		</tr>
	</tbody>
</table>

high-throughput optogenetics experiments in yeast using the automated platform lustro

光遗传学通过利用基因编码的光敏蛋白提供对细胞行为的精确控制。然而，优化这些系统以实现所需的功能通常需要多个设计-构建-测试周期，这可能既耗时又费力。为了应对这一挑战，我们开发了 Lustro，这是一个将光刺激与实验室自动化相结合的平台，能够对光遗传学系统进行高效的高通量筛选和表征。
Lustro 使用配备照明装置、摇晃装置和读板器的自动化工作站。通过使用机械臂，Lustro 使微孔板在这些设备之间自动移动，从而可以刺激光遗传学菌株并测量其反应。该协议提供了使用Lustro表征用于萌 芽酵母酿酒酵母中基因表达控制的光遗传学系统的分步指南。该协议涵盖了 Lustro 组件的设置，包括照明设备与自动化工作站的集成。它还提供了对照明设备、读板机和机器人进行编程的详细说明，确保在整个实验过程中顺利运行和数据采集。

图4A 显示了表达荧光报告基因的光遗传学菌株随时间变化的荧光值，该荧光报告基因由光诱导分裂转录因子控制。实验中使用的不同光照条件通过占空比的变化来反映，占空比表示灯亮起的时间百分比。观察到总荧光水平与光刺激的占空比成正比。 图4B 显示了同一实验的相应OD700 值。不同光照条件下光密度读数的一致性表明，该实验技术不会?...

Watch this Scientific Journal Video about Advancing Optogenetics Research Using Lustro at JoVE.com

作者聚焦:使用 Lustro 推进光遗传学研究 

该协议概述了利用自动化平台Lustro对酵母中的光遗传学系统进行高通量表征的步骤。

使用自动化平台 Lustro 在酵母中进行高通量光遗传学实验

Optogenetics offers precise control over cellular behavior by utilizing genetically encoded light-sensitive proteins. However, optimizing these systems to ...

目前有不同的设备，如生物反应器和微孔板照明器。生物反应器提供良好的时间分辨率，但通量有限。微孔板设备提供更高的通量，但缺乏自动化，无法随时间推移可靠地记录输出。Lustro 为光遗传学系统提供可靠和高通量的实时表征。它还可以与液处理机器人可以执行的检测连接。光遗传学系统是一种有吸引力的工具，因为它们对细胞行为提供了精细的空间和时间控制。更快地设计、构建和测试光遗传学系统的能力将为更好地理解和控制生物行为铺平道路。

high-throughput-optogenetics-experiments-yeast-using-automated

Research

JoVE Journal

Bioengineering

High-Throughput Optogenetics Experiments in Yeast Using the Automated Platform Lustro

589 次观看.  University of Wisconsin-Madison. 目前有不同的设备，如生物反应器和微孔板照明器。生物反应器提供良好的时间分辨率，但通量有限。微孔板设备提供更高的通量，但缺乏自动化，无法随时间推移可靠地记录输出。Lustro 为光遗传学系统提供可靠和高通量的实时表征。它还可以与液处理机器人可以执行的检测连接。光遗传学系统是一种有吸引力的工具，因为它们对细胞行为提供了精细的空间和时间控制...

视频: 作者聚焦:使用 Lustro 推进光遗传学研究 

High-throughput Protein Expression Generator Using a Microfluidic Platform

Rapidly increasing fields, such as systems biology, require the development and implementation of new technologies, enabling high-throughput and high-fidelity measurements of large systems. Microfluidics promises to fulfill many of these requirements, such as performing high-throughput screening experiments on-chip, encompassing biochemical, biophysical, and cell-based assays1. Since the early days of microfluidics devices, this field has drastically evolved, leading to the development of microfluidic large-scale integration2,3. This technology allows for the integration of thousands of micromechanical valves on a single device with a postage-sized footprint (Figure 1). We have developed a high-throughput microfluidic platform for generating in vitro expression of protein arrays (Figure 2) named PING (Protein Interaction Network Generator). These arrays can serve as a template for many experiments such as protein-protein 4, protein-RNA5 or protein-DNA6 interactions.
The device consist of thousands of reaction chambers, which are individually programmed using a microarrayer. Aligning of these printed microarrays to microfluidics devices programs each chamber with a single spot eliminating potential contamination or cross-reactivity Moreover, generating microarrays using standard microarray spotting techniques is also very modular, allowing for the arraying of proteins7, DNA8, small molecules, and even colloidal suspensions. The potential impact of microfluidics on biological sciences is significant. A number of microfluidics based assays have already provided novel insights into the structure and function of biological systems, and the field of microfluidics will continue to impact biology.

We present a microfluidic approach for the expression of protein arrays. The device consists of thousands of reaction chambers controlled by micro-mechanical valves. The microfluidic device is mated to a microarray-printed gene library. These genes are then transcribed and translated on-chip, resulting in a protein array ready for experimental use.

利用微流控平台的高通量蛋白质表达发生器

Rapidly increasing fields, such as systems biology, require the development and implementation of new technologies, enabling high-throughput and ...

科研

生物工程

High-throughput, Automated Extraction of DNA and RNA from Clinical Samples using TruTip Technology on Common Liquid Handling Robots

TruTip is a simple nucleic acid extraction technology whereby a porous, monolithic binding matrix is inserted into a pipette tip. The geometry of the monolith can be adapted for specific pipette tips ranging in volume from 1.0 to 5.0 ml. The large porosity of the monolith enables viscous or complex samples to readily pass through it with minimal fluidic backpressure. Bi-directional flow maximizes residence time between the monolith and sample, and enables large sample volumes to be processed within a single TruTip. The fundamental steps, irrespective of sample volume or TruTip geometry, include cell lysis, nucleic acid binding to the inner pores of the TruTip monolith, washing away unbound sample components and lysis buffers, and eluting purified and concentrated nucleic acids into an appropriate buffer. The attributes and adaptability of TruTip are demonstrated in three automated clinical sample processing protocols using an Eppendorf epMotion 5070, Hamilton STAR and STARplus liquid handling robots, including RNA isolation from nasopharyngeal aspirate, genomic DNA isolation from whole blood, and fetal DNA extraction and enrichment from large volumes of maternal plasma (respectively).

TruTip is a simple nucleic acid extraction technology whereby a porous, monolithic binding matrix is inserted into a pipette tip. Consequently, the sample preparation format is compatible with most liquid handling instruments, and can be used for many medium to high-throughput clinical applications and sample types.

高通量，自动化的DNA和RNA的提取从临床样品使用TruTip技术常见的液体处理机器人

TruTip is a simple nucleic acid extraction  technology whereby a porous, monolithic binding matrix is inserted into a  pipette tip. The geometry of the ...

A Versatile Automated Platform for Micro-scale Cell Stimulation Experiments

Study of cells in culture (in vitro analysis) has provided important insight into complex biological systems. Conventional methods and equipment for in vitro analysis are well suited to study of large numbers of cells (&ge;105) in milliliter-scale volumes (&ge;0.1 ml). However, there are many instances in which it is necessary or desirable to scale down culture size to reduce consumption of the cells of interest and/or reagents required for their culture, stimulation, or processing. Unfortunately, conventional approaches do not support precise and reproducible manipulation of micro-scale cultures, and the microfluidics-based automated systems currently available are too complex and specialized for routine use by most laboratories. To address this problem, we have developed a simple and versatile technology platform for automated culture, stimulation, and recovery of small populations of cells (100 - 2,000 cells) in micro-scale volumes (1 - 20 &mu;l). The platform consists of a set of fibronectin-coated microcapillaries ("cell perfusion chambers"), within which micro-scale cultures are established, maintained, and stimulated; a digital microfluidics (DMF) device outfitted with "transfer" microcapillaries ("central hub"), which routes cells and reagents to and from the perfusion chambers; a high-precision syringe pump, which powers transport of materials between the perfusion chambers and the central hub; and an electronic interface that provides control over transport of materials, which is coordinated and automated via pre-determined scripts. As an example, we used the platform to facilitate study of transcriptional responses elicited in immune cells upon challenge with bacteria. Use of the platform enabled us to reduce consumption of cells and reagents, minimize experiment-to-experiment variability, and re-direct hands-on labor. Given the advantages that it confers, as well as its accessibility and versatility, our platform should find use in a wide variety of laboratories and applications, and prove especially useful in facilitating analysis of cells and stimuli that are available in only limited quantities.

We have developed an automated cell culture and interrogation platform for micro-scale cell stimulation experiments. The platform offers simple, versatile, and precise control in cultivating and stimulating small populations of cells, and recovering lysates for molecular analyses. The platform is well suited to studies that use precious cells and/or reagents.

一种多功能自动化平台微尺度细胞刺激实验

Study of cells in culture (in vitro analysis) has provided important insight into complex biological systems. Conventional methods and equipment for in ...

CometChip: A High-throughput 96-Well Platform for Measuring DNA Damage in Microarrayed Human Cells

DNA damaging agents can promote aging, disease and cancer and they are ubiquitous in the environment and produced within human cells as normal cellular metabolites. Ironically, at high doses DNA damaging agents are also used to treat cancer. The ability to quantify DNA damage responses is thus critical in the public health, pharmaceutical and clinical domains. Here, we describe a novel platform that exploits microfabrication techniques to pattern cells in a fixed microarray. The &#8216;CometChip&#8217; is based upon the well-established single cell gel electrophoresis assay (a.k.a. the comet assay), which estimates the level of DNA damage by evaluating the extent of DNA migration through a matrix in an electrical field. The type of damage measured by this assay includes abasic sites, crosslinks, and strand breaks. Instead of being randomly dispersed in agarose in the traditional assay, cells are captured into an agarose microwell array by gravity. The platform also expands from the size of a standard microscope slide to a 96-well format, enabling parallel processing. Here we describe the protocols of using the chip to evaluate DNA damage caused by known genotoxic agents and the cellular repair response followed after exposure. Through the integration of biological and engineering principles, this method potentiates robust and sensitive measurements of DNA damage in human cells and provides the necessary throughput for genotoxicity testing, drug development, epidemiological studies and clinical assays.

We describe here a platform that allows comet assay detection of DNA damage with unprecedented throughput. The device patterns mammalian cells into a microarray and enables parallel processing of 96 samples. The approach facilitates analysis of base level DNA damage, exposure-induced DNA damage and DNA repair kinetics.

CometChip:一种高通量的96孔平台的微阵列人类细胞测量DNA损伤

DNA damaging agents can promote aging, disease and cancer and they are ubiquitous in the environment and produced within human cells as normal cellular ...

Using Tomoauto: A Protocol for High-throughput Automated Cryo-electron Tomography

Cryo-electron tomography (Cryo-ET) is a powerful three-dimensional (3-D) imaging technique for visualizing macromolecular complexes in their native context at a molecular level. The technique involves initially preserving the sample in its native state by rapidly freezing the specimen in vitreous ice, then collecting a series of micrographs from different angles at high magnification, and finally computationally reconstructing a 3-D density map. The frozen-hydrated specimen is extremely sensitive to the electron beam and so micrographs are collected at very low electron doses to limit the radiation damage. As a result, the raw cryo-tomogram has a very low signal to noise ratio characterized by an intrinsically noisy image. To better visualize subjects of interest, conventional imaging analysis and sub-tomogram averaging in which sub-tomograms of the subject are extracted from the initial tomogram and aligned and averaged are utilized to improve both contrast and resolution. Large datasets of tilt-series are essential to understanding and resolving the complexes at different states, conditions, or mutations as well as obtaining a large enough collection of sub-tomograms for averaging and classification. Collecting and processing this data can be a major obstacle preventing further analysis. Here we describe a high-throughput cryo-ET protocol based on a computer-controlled 300kV cryo-electron microscope, a direct detection device (DDD) camera and a highly effective, semi-automated image-processing pipeline software wrapper library tomoauto developed in-house. This protocol has been effectively utilized to visualize the intact type III secretion system (T3SS) in Shigella flexneri minicells. It can be applicable to any project suitable for cryo-ET.

We present a protocol on how to utilize high-throughput cryo-electron tomography to determine high resolution in situ structures of molecular machines. The protocol permits large amounts of data to be processed, avoids common bottlenecks and reduces resource downtime, allowing the user to focus on important biological questions.

使用Tomoauto:一个协议，用于高通量自动化低温电子断层扫描

Cryo-electron tomography (Cryo-ET) is a powerful three-dimensional (3-D) imaging technique for visualizing macromolecular complexes in their native ...

A Microfluidic Platform for High-throughput Single-cell Isolation and Culture

Studying the heterogeneity of single cells is crucial for many biological questions, but is technically difficult. Thus, there is a need for a simple, yet high-throughput, method to perform single-cell culture experiments. Here, we report a microfluidic chip-based strategy for high-efficiency single-cell isolation (~77%) and demonstrate its capability of performing long-term single-cell culture (up to 7 d) and cellular heterogeneity analysis using clonogenic assay. These applications were demonstrated with KT98 mouse neural stem cells, and A549 and MDA-MB-435 human cancer cells. High single-cell isolation efficiency and long-term culture capability are achieved by using different sizes of microwells on the top and bottom of the microfluidic channel. The small microwell array is designed for precisely isolating single-cells, and the large microwell array is used for single-cell clonal culture in the microfluidic chip. This microfluidic platform constitutes an attractive approach for single-cell culture applications, due to its flexibility of adjustable cell culture spaces for different culture strategies, without decreasing isolation efficiency.

Here, we present a protocol for isolating and culturing single cells with a microfluidic platform, which utilizes a new microwell design concept to allow for high-efficiency single cell isolation and long-term clonal culture.

高通量单细胞的分离和文化的微流体平台

Studying the heterogeneity of single cells is crucial for many biological questions, but is technically difficult. Thus, there is a need for a simple, yet ...

A High-throughput Cell Microarray Platform for Correlative Analysis of Cell Differentiation and Traction Forces

Microfabricated cellular microarrays, which consist of contact-printed combinations of biomolecules on an elastic hydrogel surface, provide a tightly controlled, high-throughput engineered system for measuring the impact of arrayed biochemical signals on cell differentiation. Recent efforts using cell microarrays have demonstrated their utility for combinatorial studies in which many microenvironmental factors are presented in parallel. However, these efforts have focused primarily on investigating the effects of biochemical cues on cell responses. Here, we present a cell microarray platform with tunable material properties for evaluating both cell differentiation by immunofluorescence and biomechanical cell&#8211;substrate interactions by traction force microscopy. To do so, we have developed two different formats utilizing polyacrylamide hydrogels of varying Young&#39;s modulus fabricated on either microscope slides or glass-bottom Petri dishes. We provide best practices and troubleshooting for the fabrication of microarrays on these hydrogel substrates, the subsequent cell culture on microarrays, and the acquisition of data. This platform is well-suited for use in investigations of biological processes for which both biochemical (e.g., extracellular matrix composition) and biophysical (e.g., substrate stiffness) cues may play significant, intersecting roles.

Cell differentiation is regulated by a host of microenvironmental factors, including both matrix composition and substrate material properties. We describe here a technique utilizing cell microarrays in conjunction with traction force microscopy to evaluate both cell differentiation and biomechanical cell&#8211;substrate interactions as a function of microenvironmental context.

细胞分化和牵引力的相关分析高通量细胞芯片平台

Microfabricated cellular microarrays, which consist of contact-printed combinations of biomolecules on an elastic hydrogel surface, provide a tightly ...

Use of High-Throughput Automated Microbioreactor System for Production of Model IgG1 in CHO Cells

Automated microscale bioreactors (15 mL) can be a useful tool for cell culture engineers. They facilitate the simultaneous execution of a wide variety of experimental conditions while minimizing potential process variability. Applications of this approach include: clone screening, temperature and pH shifts,&#160;media and supplement optimization. Furthermore, the small reactor volumes are conducive to large Design of Experiments that investigate a wide range of conditions. This allows upstream processes to be significantly optimized before scale-up where experimentation is more limited in scope due to time and economic constraints. Automated microscale bioreactor systems offer various advantages over traditional small scale cell culture units, such as shake flasks or spinner flasks. However, during pilot scale process development&#160;significant care must be taken to ensure that these advantages are realized. When run with care, the system can enable high level automation, can be programmed to run DOE&#39;s with a higher number of variables&#160;and can reduce sampling time when integrated with a nutrient analyzer or cell counter. Integration of the expert-derived heuristics presented here, with current automated microscale bioreactor experiments can minimize common pitfalls that hinder meaningful results. In the extreme, failure to adhere to the principles laid out here can lead to equipment damage that requires expensive repairs. Furthermore, the microbioreactor systems have small culture volumes&#160;making characterization of cell culture conditions difficult. The number and amount of samples taken in-process in batch mode culture is limited as operating volumes cannot fall below 10 mL. This method will discuss the benefits and drawbacks of microscale bioreactor systems.

A detailed protocol for the concurrent operation of 48 parallel cell cultures under varied conditions in a microbioreactor system is presented. Cell culture process, harvest and subsequent antibody titer analysis are described.

高通量自动 Microbioreactor 系统在 CHO 细胞模型 IgG1 生产中的应用

Automated microscale bioreactors (15 mL) can be a useful tool for cell culture engineers. They facilitate the simultaneous execution of a wide variety of ...

Process Optimization using High Throughput Automated Micro-Bioreactors in Chinese Hamster Ovary Cell Cultivation

Optimization of bioprocesses to increase the yield of desired products is of importance in the biopharmaceutical industry. This can be achieved by strain selection and by developing bioprocess parameters. Shake flasks have been used for this purpose. They, however, lack the capability to control the process parameters such as pH and dissolved oxygen (DO). This limitation can be overcome with the help of an automated micro-bioreactor. These bioreactors mimic cultivation at a larger scale. One of the major advantages of this system is the integration of the Design of Experiment (DOE) in the software. This integration enables establishing a design where multiple process parameters can be varied simultaneously. The critical process parameters and optimum bioprocess conditions can be analyzed within the software. The focus of the work presented here is to introduce the user to the steps involved in process design in the software and incorporation of the DOE within the cultivation run.

Here, we present a detailed procedure to run a Design of Experiment in an automated micro-bioreactor followed by cell harvest and protein quantification using a Protein A column.

中国仓鼠卵巢细胞培养中高通量自动化微生物反应器工艺优化

Optimization of bioprocesses to increase the yield of desired products is of importance in the biopharmaceutical industry. This can be achieved by strain ...

High Throughput Yeast Strain Phenotyping with Droplet-Based RNA Sequencing

The powerful tools available to edit yeast genomes have made this microbe a valuable platform for engineering. While it is now possible to construct libraries of millions of genetically distinct strains, screening for a desired phenotype remains a significant obstacle. With existing screening techniques, there is a tradeoff between information output and throughput, with high-throughput screening typically being performed on one product of interest. Therefore, we present an approach to accelerate strain screening by adapting single cell RNA sequencing to isogenic picoliter colonies of genetically engineered yeast strains. To address the unique challenges of performing RNA sequencing on yeast cells, we culture isogenic yeast colonies within hydrogels and spheroplast prior to performing RNA sequencing. The RNA sequencing data can be used to infer yeast phenotypes and sort out engineered pathways. The scalability of our method addresses a critical obstruction in microbial engineering.

A bottleneck in the &#8216;design-build-test&#8217; cycle of microbial engineering is the speed at which we can perform functional screens of strains. We describe a high-throughput method for strain screening applied to hundreds to thousands of yeast cells per experiment that utilizes droplet-based RNA sequencing.

高通量酵母菌株，带基于滴滴的RNA测序

The powerful tools available to edit yeast genomes have made this microbe a valuable platform for engineering. While it is now possible to construct ...