<html>在这里，我们讨论的是在卢卡斯[1-3]的实验程序。为了说明卢卡斯的概念证明，我们将描述为一个整体的血液样本的成像过程。 答：成像设置卢卡斯成像平台表现出显着的优势，对现有的护理点流式细胞仪和医疗诊断工具，尤其是对资源有限的环境，提供成本效益和紧凑的替代。检测细胞的形象，而不是卢卡斯，而不是捕捉的是从每个单元格的背景光的散射光的干扰创建细胞的数字全息图。仔细控制部分空间相干性的照明是至关重要的，使全息记录。 1。数字传感器阵列卢卡斯平台采用光电传感器阵列，以数字方式记录单个细胞的全息图。为此，收取夫妇器件（CCD;示例模型：KAI - 11002，从柯达KAF - 39000，）或互补金属氧化物半导体芯片（CMOS，样品型号：MT9P031，美光科技公司），都可以使用。像素大小的柯达控告夫妇设备，开，KAF - 39000 11002，微米CMOS图像传感器的9微米，6.8微米和2.2微米，与一个积极的视场为10平方厘米​​，18平方厘米，24.4 mm2的分别。 [1-2]。 2。光源不像大多数其他的显微成像方式，卢卡斯不需要激光，因此即使一个简单的发光二极管（LED）可用于照明。为了使可调谐波长照明，我们也可以利用与标准级熔融石英纤维组成的纤维束（77564，新港）和针孔与氙气灯（T260基石，新港公司）的单色~~位于〜5-10厘米以上的传感器表面的直径为100微米。这种可调谐波长照明配置提供了一个灵活的平台，可以调节细胞的全息签名，并可以合成混合数字签名，以改善信噪比更好的表征的准确性和特异性。 [3] B.样品制备和成像将演示一个基于片内图像卢卡斯的概念证明，使用一个异构的解决方案，如下所述。类似的协议，可应用于各种其他类型的细胞[1-3]。 1。全血稀释的异构解决方案的准备<ol><li>要开始的过程，获得一个整体的血液样本和各种直径（5，10和20微米。杜克科学）的聚苯乙烯微球，并把他们全部室温。 </li><li>添加到稀释液5毫升无菌聚丙烯管2毫升的RPMI 1640液体培养基（Fisher Scientific则）。 </li><li>全血样品孵育30分钟后，移液器10μL沉淀红细胞标本，并转移到RPMI解决方案。 </li><li>添加到红细胞的RPMI稀释一个总体积20μL的聚苯乙烯微球，并轻轻吹打，或用搅拌棒使用磁力搅拌器搅拌单元解决方案。 </li><li>放置到一个涵盖5-15μL的异构解决方案的总体积滑和地点，在解决方案的第二个相同的覆盖使用镊子滑移。试样应夹在两者之间的盖玻片均匀。 </li><li>然后，使用真空笔（NT57 - 636，埃德蒙光学）加载到的图像传感器阵列的活跃地区的样本。 </li></ol> 2。全血染色<ol><li> 0.1％的混合物准备开始缓冲曙红Y和稀释后的新亚甲基解决方案，通过使用功率型纯曙红Y（MW = 691.85，Acros Organics公司），纯锌的新亚甲基蓝染料（MW = 347.90，Acros Organics公司）草酸钾一水（99.0％试剂ACS，Acros Organics公司）;每一个解决方案是0.45微米孔径Syringless过滤器（滤纸）水的白血细胞染色与纯化。 </li><li>然后，轻轻搅动一个溶于水染色试剂全血标本在聚丙烯烧杯1:1的体积比使用磁力搅拌2分钟，孵育10分钟。 </li><li> 5 mL聚丙烯管转移到一个沾满血的解决方案和50μL的RPMI 1640溶液1.95毫升（尔科技）稀释收购体积比为1:200。然后，涡稀释为30秒。也可以在此阶段引入一个不同的稀释率。 </li><li>移液器染色细胞的10-100μL盖玻片之间，或在一个微水库解决方案。然后，使用真空笔（NT57 - 636，埃德蒙光学），地方上的传感器阵列的活跃地区的标本。 </li></ol>代表性的成果ve_content“&gt; <img src="/files/ftp_upload/1650/1650Fig1tmb.jpg" alt="图1" border="0" /> 图1：卢卡斯可以准确计数和各种细胞和微型物体的二维全息签名，而无需使用任何镜头，激光或其他笨重的光学元件为基础的数字分化。在这个数字说明特色卢卡斯各种微型对象的签名和对传统的显微镜与40X物镜获得的图像进行比较。请<a href="https://www.jove.com/files/ftp_upload/1650/1650Fig1.jpg">点击这里</a>看到图1的放大版本。 <img src="/files/ftp_upload/1650/1650Fig2.jpg" alt="图2" /> 图2：（左）原始图像的异构混合物含有红细胞，10um的，5um的和3微米的颗粒。 （右）全自动卢卡斯相同的视野表征结果说明。注意决策算法是强大的高密度地区以及颗粒低信号特征信噪比3um珠等。 <img src="/files/ftp_upload/1650/1650Fig3tmb.jpg" alt="图3" border="0" /> 图3：卢卡斯自定义接口的说明。基于Java的卢卡斯软件允许，如传感器像素大小或光的波长不同实验条件下的输入。也可以选择一个图像的特定领域和靶细胞的模式可以由用户定义，建立一个统计细胞阴影库。收购了卢卡斯的形象，然后可以基于这种训练数据（即细胞阴影库）和标记（计）图像显示给用户的特点。计数结果的统计数据也将存储作为一个XML文件，作进一步的分析。

<ol>
	<li>Seo, S., Su, T., Tseng, D. K., Erlinger, A., Ozcan, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term="Lensfree+Holographic+Imaging+for+On-Chip+Cytometry+and+Diagnostics".">"Lensfree Holographic Imaging for On-Chip Cytometry and Diagnostics".</a> Lab Chip. , (2009).</li><li>Su, T., Seo, S., Erlinger, A., Ozcan, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term="High-throughput+Imaging+and+Characterization+of+a+Heterogeneous+Cell+Solution+on+a+Chip"+.">"High-throughput Imaging and Characterization of a Heterogeneous Cell Solution on a Chip" .</a> Biotechnology and Bioengineering. , (2008).</li><li>Su, T., Seo, S., Erlinger, A., Ozcan, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term="Multi-color+LUCAS:+Lensfree+on-chip+cytometry+using+tunable+monochromatic+illumination+and+digital+noise+reduction".">"Multi-color LUCAS: Lensfree on-chip cytometry using tunable monochromatic illumination and digital noise reduction".</a> Cellular and Molecular Bioengineering. , (2008).</li></ol>

我们已经说明，卢卡斯平台可以准确计数和基于全息签名的芯片识别各种micro-objects/cells，点的医疗诊断和高通量细胞生物学提供了一种有前途的工具。为了准确地处理所记录的全息图案，我们实现了一个自定义开发卢卡斯决定的软件。这种算法，它采用了统计培训卢卡斯图像所造成的衍射图像数据库，确定细胞内的异构解决方案的各种功能，单个细胞的类型和相对位置进行分类和应用阈值后，放...

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lensless on-chip imaging of cells provides a new tool for high-throughput cell-biology and medical diagnostics

工作与其他镜头和光学组件的物镜使用常规的光学显微镜图像细胞。这种传统的方法虽然很有效，有一定的局限性，成像平台，使之与先进国家的最先进的微流体兼容的小型化。在这份报告中，我们引入一个无透镜片上的成像的概念称为卢卡斯的实验细节（<strong<em&gt; L</em</strong&gt; ensless<strong<em&gt; U</em</strong&gt; LTRA宽领域的视图<strong<em&gt; C</em</strong&gt; ELL监测<strong<em&gt; A</em</strong&gt; rray平台的基础上<strong<em&gt; S</em</strong&gt;哈多成像），不需要任何显微镜目标或其他笨重的光学元件的图像通过一个超宽的领域可以跨越大如一个异构的细胞解决方案〜18厘米<sup&gt; 2</sup&gt;。此外，不同于传统的显微镜，卢卡斯可以形象的利益异质细胞多〜5毫米的深度场的解决方案，而不需要重新聚焦对应〜9 mL样品量。这种成像平台记录的每一个细胞，其视野内的兴趣和自动数字处理这些细胞阴影的阴影（即，数字全息图无透镜），可确定的类型，数量和细胞内的解决方案的相对位置。因为它不需要任何笨重的光学元件或机械扫描阶段，它提供了一个显着小型化的平台，同时降低了成本，这一点非常重要的护理诊断工具，尤其是点。此外，这个平台的成像吞吐量订单数量级比传统的光学显微镜，高通量细胞生物学实验，这可能是极为宝贵的更好。

Watch this Scientific Journal Video about Lensless On-chip Imaging of Cells Provides a New Tool for High-throughput Cell-Biology and Medical Diagnostics at JoVE.com

Lensless On-chip Imaging of Cells Provides a New Tool for High-throughput Cell-Biology and Medical Diagnostics

Lensfree片上的成像和细胞的特性说明。这片上的细胞成像方法用于医疗诊断和高通量细胞生物学的应用提供了一个紧凑和成本效益的工具，使得它特别适合资源贫乏地区。

无透镜片上的细胞成像提供了一种高通量细胞生物学和医疗诊断的新工具

Conventional optical microscopes image cells by use of objective lenses that work together with other lenses and optical components. While quite ...

lensless-on-chip-imaging-cells-provides-new-tool-for-high-throughput

Research

JoVE Journal

Biology

11.9K 次观看.  University of California, Los Angeles. Lensfree片上的成像和细胞的特性说明。这片上的细胞成像方法用于医疗诊断和高通量细胞生物学的应用提供了一个紧凑和成本效益的工具，使得它特别适合资源贫乏地区。

视频: Lensless On-chip Imaging of Cells Provides a New Tool for High-throughput Cell-Biology and Medical Diagnostics

Tracheotomy: A Method for Transplantation of Stem Cells to the Lung

Lung disease is a leading cause of death and likely to become an epidemic given increases in pollution and smoking worldwide. Advances in stem cell therapy may alleviate many of the symptoms associated with lung disease and induce alveolar repair in adults. Concurrent with the ongoing search for stem cells applicable for human treatment, precise delivery and homing (to the site of disease) must be reassured for successful therapy. Here, I report that stem cells can safely be instilled via the trachea opening a non-stop route to the lung. This method involves a skin incision, caudal insertion of a cannula into and along the tracheal lumen, and injection of a stem cell vehicle mixture into airways of the lung. A broad range of media solutions and stabilizers can be instilled via tracheotomy, resulting in the ability to deliver a wider range of cell types. With alveolar epithelium confining these cells to the lumen, lung expansion and negative pressure during inhalation may also assist in stem cell integration. Tracheal delivery of stem cells, with a quick uptake and the ability to handle a large range of treatments, could accelerate the development of cell-based therapies, opening new avenues for treatment of lung disease.

Significant breakthroughs in stem cell identification are continuously being made. To translate these discoveries, however, novel methods for cellular delivery must be devised. Here I report that the airways provide a safe route for stem cell transplantation to the lungs.

气管切开术：干细胞移植肺的方法

Lung disease is a leading cause of death and likely to become an epidemic given increases in pollution and smoking worldwide. Advances in stem cell ...

科研

生物学

A Gradient-generating Microfluidic Device for Cell Biology

 The fabrication and operation of a gradient-generating microfluidic device for studying cellular behavior is described.  A microfluidic platform is an enabling experimental tool, because it can precisely manipulate fluid flows, enable high-throughput experiments, and generate stable soluble concentration gradients.  Compared to conventional gradient generators, poly(dimethylsiloxane) (PDMS)-based  microfluidic devices can generate stable concentration gradients of growth factors with well-defined profiles.  Here, we developed simple gradient-generating microfluidic devices with three separate inlets.  Three microchannels combined into one microchannel to generate concentration gradients.  The stability and shape of growth factor gradients were confirmed by fluorescein isothyiocyanate (FITC)-dextran with a molecular weight similar to epidermal growth factor (EGF).  Using this microfluidic device, we demonstrated that fibroblasts exposed to concentration gradients of EGF migrated toward higher concentrations.  The directional orientation of cell migration and motility of migrating cells were quantitatively assessed by cell tracking analysis.  Thus, this gradient-generating microfluidic device might be useful for studying and analyzing the behavior of migrating cells.

We describe a protocol for the microfabrication of the gradient-generating microfluidic device that can generate spatial and temporal gradients in well-defined microenvironment. In this approach, the gradient-generating microfluidic device can be used to study directed cell migration, embryogenesis, wound healing, and cancer metastasis.

一个渐变产生的微流体装置，细胞生物学

 The fabrication and operation of a gradient-generating microfluidic device for studying cellular behavior is described.  A microfluidic platform is an ...

Biochemical and High Throughput Microscopic Assessment of Fat Mass in Caenorhabditis Elegans

The nematode C. elegans has emerged as an important model for the study of conserved genetic pathways regulating fat metabolism as it relates to human obesity and its associated pathologies. Several previous methodologies developed for the visualization of C. elegans triglyceride-rich fat stores have proven to be erroneous, highlighting cellular compartments other than lipid droplets. Other methods require specialized equipment, are time-consuming, or yield inconsistent results. We introduce a rapid, reproducible, fixative-based Nile red staining method for the accurate and rapid detection of neutral lipid droplets in C. elegans. A short fixation step in 40% isopropanol makes animals completely permeable to Nile red, which is then used to stain animals. Spectral properties of this lipophilic dye allow it to strongly and selectively fluoresce in the yellow-green spectrum only when in a lipid-rich environment, but not in more polar environments. Thus, lipid droplets can be visualized on a fluorescent microscope equipped with simple GFP imaging capability after only a brief Nile red staining step in isopropanol. The speed, affordability, and reproducibility of this protocol make it ideally suited for high throughput screens. We also demonstrate a paired method for the biochemical determination of triglycerides and phospholipids using gas chromatography mass-spectrometry. This more rigorous protocol should be used as confirmation of results obtained from the Nile red microscopic lipid determination. We anticipate that these techniques will become new standards in the field of C. elegans metabolic research.

Biochemical and High Throughput Microscopic Assessment of Fat Mass in Caenorhabditis Elegans

We present robust biochemical and microscopic methods for studying Caenorhabditis elegans lipid stores. A rapid, simple, fixing-staining procedure for fluorescent lipid droplet imaging leverages the spectral properties of the lipophilic dye Nile red. We then present biochemical measurement of triglycerides and phospholipids using solid phase extraction and gas chromatography-mass spectrometry.

生化和高通量的微观评估的脂肪含量<em&gt;秀丽隐杆线虫</em

The nematode C. elegans has emerged as an important  model for the study of conserved genetic pathways regulating fat metabolism as  it relates to human ...

Anti-Nuclear Antibody Screening Using HEp-2 Cells

The American College of Rheumatology position statement on ANA testing stipulates the use of IIF as the gold standard method for ANA screening1. Although IIF is an excellent screening test in expert hands, the technical difficulties of processing and reading IIF slides &#8211; such as the labor intensive slide processing, manual reading, the need for experienced, trained technologists and the use of dark room &#8211; make the IIF method difficult to fit in the workflow of modern, automated laboratories.
The first and crucial step towards high quality ANA screening is careful slide processing. This procedure is labor intensive, and requires full understanding of the process, as well as attention to details and experience.
Slide reading is performed by fluorescent microscopy in dark rooms, and is done by trained technologists who are familiar with the various patterns, in the context of cell cycle and the morphology of interphase and dividing cells. Provided that IIF is the first line screening tool for SARD, understanding the steps to correctly perform this technique is critical.
Recently, digital imaging systems have been developed for the automated reading of IIF slides. These systems, such as the NOVA View Automated Fluorescent Microscope, are designed to streamline the routine IIF workflow. NOVA View acquires and stores high resolution digital images of the wells, thereby separating image acquisition from interpretation; images are viewed an interpreted on high resolution computer monitors. It stores images for future reference and supports the operator&#8217;s interpretation by providing fluorescent light intensity data on the images. It also preliminarily categorizes results as positive or negative, and provides pattern recognition for positive samples. In summary, it eliminates the need for darkroom, and automates and streamlines the IIF reading/interpretation workflow. Most importantly, it increases consistency between readers and readings. Moreover, with the use of barcoded slides, transcription errors are eliminated by providing sample traceability and positive patient identification. This results in increased patient data integrity and safety.
The overall goal of this video is to demonstrate the IIF procedure, including slide processing, identification of common IIF patterns, and the introduction of new advancements to simplify and harmonize this technique.

Indirect immunofluorescent (IIF) assays have traditionally been used for the detection of antinuclear antibodies (ANA) in human serum. The presence of these antibodies can aid in the diagnosis of systemic autoimmune rheumatic diseases (SARD). This protocol demonstrates how to effectively perform the IIF technique to accurately detect these autoantibodies.

抗核抗体筛选使用Hep-2细胞

The American College of Rheumatology position statement on ANA testing stipulates the use of IIF as the gold standard method for ANA screening1. Although ...

High-throughput Image Analysis of Tumor Spheroids: A User-friendly Software Application to Measure the Size of Spheroids Automatically and Accurately

The increasing number of applications of three-dimensional (3D) tumor spheroids as an in vitro model for drug discovery requires their adaptation to large-scale screening formats in every step of a drug screen, including large-scale image analysis. Currently there is no ready-to-use and free image analysis software to meet this large-scale format. Most existing methods involve manually drawing the length and width of the imaged 3D spheroids, which is a tedious and time-consuming process. This study presents a high-throughput image analysis software application &#8211; SpheroidSizer, which measures the major and minor axial length of the imaged 3D tumor spheroids automatically and accurately; calculates the volume of each individual 3D tumor spheroid; then outputs the results in two different forms in spreadsheets for easy manipulations in the subsequent data analysis. The main advantage of this software is its powerful image analysis application that is adapted for large numbers of images. It provides high-throughput computation and quality-control workflow. The estimated time to process 1,000 images is about 15 min&#160;on a minimally configured laptop, or around 1 min&#160;on a multi-core performance workstation. The graphical user interface (GUI) is also designed for easy quality control, and users can manually override the computer results. The key method used in this software is adapted from the active contour algorithm, also known as Snakes, which is especially suitable for images with uneven illumination and noisy background that often plagues automated imaging processing in high-throughput screens. The complimentary &#8220;Manual Initialize&#8221; and &#8220;Hand Draw&#8221; tools provide the flexibility to SpheroidSizer in dealing with various types of spheroids and diverse quality images. This high-throughput image analysis software remarkably reduces labor and speeds up the analysis process. Implementing this software is beneficial for 3D tumor spheroids to become a routine in vitro model for drug screens in industry and academia.

We present a high-throughput image analysis software application to measure the size of three-dimensional tumor spheroids imaged with bright-field microscopy. This application provides a fast and effective way to examine the effects of therapeutic drugs on spheroids, which is beneficial for researchers who wish to use spheroids in drug screens.

肿瘤细胞球体的高通量图像分析：一个用户友好的软件应用来测量球体的大小自动准确

The increasing number of applications of three-dimensional (3D) tumor spheroids as an in vitro model for drug discovery requires their adaptation to ...

High-throughput Screening for Chemical Modulators of Post-transcriptionally Regulated Genes

Both transcriptional and post-transcriptional regulation have a profound impact on genes expression. However, commonly adopted cell-based screening assays focus on transcriptional regulation, being essentially aimed at the identification of promoter-targeting molecules. As a result, post-transcriptional mechanisms are largely uncovered by gene expression targeted drug development. Here we describe a cell-based assay aimed at investigating the role of the 3&#39; untranslated region (3&#8217; UTR) in the modulation of the fate of its mRNA, and at identifying compounds able to modify it. The assay is based on the use of a luciferase reporter construct containing the 3&#8217; UTR of a gene of interest stably integrated into a disease-relevant cell line. The protocol is divided into two parts, with the initial focus on the primary screening aimed at the identification of molecules affecting luciferase activity after 24 hr of treatment. The second part of the protocol describes the counter-screening necessary to discriminate compounds modulating luciferase activity specifically through the 3&#8217; UTR. In addition to the detailed protocol and representative results, we provide important considerations about the assay development and the validation of the hit(s) on the endogenous target. The described cell-based reporter gene assay will allow scientists to identify molecules modulating protein levels via post-transcriptional mechanisms dependent on a 3&#8217; UTR.

Here we describe a cell-based reporter gene assay as a valuable tool to screen chemical libraries for compounds modulating post-transcriptional control mechanisms exerted through 3&#8217; UTR.

高通量筛选转录后调控基因的化学调节剂

Both transcriptional and post-transcriptional regulation have a profound impact on genes expression. However, commonly adopted cell-based screening assays ...

High-Throughput Metabolic Profiling for Model Refinements of Microalgae

Metabolic models are reconstructed based on an organism&#39;s available genome annotation and provide predictive tools to study metabolic processes at a systems-level. Genome-scale metabolic models may include gaps as well as reactions that are unverified experimentally. Reconstructed models of newly isolated microalgal species will result in weaknesses due to these gaps, as there is usually sparse biochemical evidence available for the metabolism of such isolates. The phenotype microarray (PM) technology is an effective, high-throughput method that functionally determines cellular metabolic activities&#160;in response to a wide array of entry metabolites. Combining the high throughput phenotypic assays with metabolic modeling can allow existing metabolic network models to be rapidly reconstructed or optimized by providing biochemical evidence to support and expand genomic evidence. This work will show the use of PM assays for the study of microalgae by using the green microalgal model species Chlamydomonas reinhardtii as an example. Experimental evidence for over 254 reactions obtained by PM was used in this study to expand and refine a genome-scale C. reinhardtii metabolic network model, iRC1080, by approximately 25 percent. The protocol created here can be used as a basis for functionally profiling the metabolism of other microalgae, including known microalgae mutants and new isolates.

This protocol demonstrates the use of a phenotype microarray (PM) technology platform to define metabolic requirements of Chlamydomonas reinhardtii, a green microalga, and refine an existing metabolic network model.

用于微藻模型改进的高通量代谢分析

Metabolic models are reconstructed based on an organism&#39;s available genome annotation and provide predictive tools to study metabolic processes at a ...

High-Throughput Live Imaging of Microcolonies to Measure Heterogeneity in Growth and Gene Expression

Precise measurements of between- and within-strain heterogeneity in microbial growth rates are essential for understanding genetic and environmental inputs into stress tolerance, pathogenicity, and other key components of fitness. This manuscript describes a microscope-based assay that tracks approximately 105&#160;Saccharomyces cerevisiae microcolonies per experiment. After automated time-lapse imaging of yeast immobilized in a multiwell plate, microcolony growth rates are easily analyzed with custom image-analysis software. For each microcolony, expression and localization of fluorescent proteins and survival of acute stress can also be monitored. This assay allows precise estimation of strains&#39; average growth rates, as well as comprehensive measurement of heterogeneity in growth, gene expression, and stress tolerance within clonal populations.

Yeast growth phenotypes are precisely measured through highly parallel time-lapse imaging of immobilized cells growing into microcolonies. Simultaneously, stress tolerance, protein expression, and protein localization can be monitored, generating integrated datasets to study how environmental and genetic differences, as well as gene-expression heterogeneity among isogenic cells, modulate growth.

微殖器的高通量实时成像，测量生长和基因表达中的异质性

Precise measurements of between- and within-strain heterogeneity in microbial growth rates are essential for understanding genetic and environmental ...

A High-Throughput Comet Assay Approach for Assessing Cellular DNA Damage

Cells are continually exposed to agents arising from the internal and external environments, which may damage DNA. This damage can cause aberrant cell function, and therefore DNA damage may play a critical role in the development of, conceivably, all major human diseases, e.g., cancer, neurodegenerative and cardiovascular disease, and aging. Single-cell gel electrophoresis (i.e., the comet assay) is one of the most common and sensitive methods to study the formation and repair of a wide range of types of DNA damage (e.g., single- and double-strand breaks, alkali-labile sites, DNA-DNA crosslinks, and, in combination with certain repair enzymes, oxidized purines, and pyrimidines), in both in vitro and in vivo systems. However, the low sample throughput of the conventional assay and laborious sample workup are limiting factors to its widest possible application. With the "scoring" of comets increasingly automated, the limitation is now the ability to process significant numbers of comet slides. Here, a high-throughput (HTP) variant of the comet assay (HTP comet assay) has been developed, which significantly increases the number of samples analyzed, decreases assay run time, the number of individual slide manipulations, reagent requirements, and risk of physical damage to the gels. Furthermore, the footprint of the electrophoresis tank is significantly decreased due to the vertical orientation of the slides and integral cooling. Also reported here is a novel approach to chilling comet assay slides, which conveniently and efficiently facilitates the solidification of the comet gels. Here, the application of these devices to representative comet assay methods has been described. These simple innovations greatly support the use of the comet assay and its application to areas of study such as exposure biology, ecotoxicology, biomonitoring, toxicity screening/testing, together with understanding pathogenesis.

The comet assay is a popular means of detecting DNA damage. This study describes an approach to running slides in representative variants of the comet assay. This approach significantly increased the number of samples while decreasing assay run-time, the number of slide manipulations, and the risk of damage to gels.

一种用于评估细胞DNA损伤的高通量彗星检测方法

Cells are continually exposed to agents arising from the internal and external environments, which may damage DNA. This damage can cause aberrant cell ...

A High-Throughput Platform for Culture and 3D Imaging of Organoids

The characterization of a large number of three-dimensional (3D) organotypic cultures (organoids) at different resolution scales is currently limited by standard imaging approaches. This protocol describes a way to prepare microfabricated organoid culture chips, which enable multiscale, 3D live imaging on a user-friendly instrument requiring minimal manipulations and capable of up to 300 organoids/h imaging throughput. These culture chips are compatible with both air and immersion objectives (air, water, oil, and silicone) and a wide range of common microscopes (e.g., spinning disk, point scanner confocal, wide field, and brightfield). Moreover, they can be used with light-sheet modalities such as the single-objective, single-plane illumination microscopy (SPIM) technology (soSPIM). 
The protocol described here gives detailed steps for the preparation of the microfabricated culture chips and the culture and staining of organoids. Only a short length of time is required to become familiar with, and consumables and equipment can be easily found in normal biolabs. Here, the 3D imaging capabilities will be demonstrated only with commercial standard microscopes (e.g., spinning disk for 3D reconstruction and wide field microscopy for routine monitoring).

This paper presents a fabrication protocol for a new type of culture substrate with hundreds of microcontainers per mm2, in which organoids can be cultured and observed using high-resolution microscopy. The cell seeding and immunostaining protocols are also detailed.

用于类器官培养和 3D 成像的高通量平台

The characterization of a large number of three-dimensional (3D) organotypic cultures (organoids) at different resolution scales is currently limited by ...