<html>ここでは、LUCAS [1-3]に関与している実験的な手順を説明します。 LUCASの概念実証を説明するために我々は、全血サンプルのイメージングプロセスを説明します。 A.イメージングセットアップ LUCASイメージングプラットフォームは、特に資源が限られた設定のために、既存のポイントオブケアサイトメトリーや医療診断ツールに費用対効果の高い、コンパクトな代替手段を提供するために重要な利点を示す。むしろ細胞の画像を検出するよりも、LUCASは、代わりに背景光との各セルからの散乱光の干渉によって作成されているセルのデジタルホログラムをキャプチャします。照明の部分的な空間的コヒーレンスの注意深い制御は、ホログラフィック記録を有効にするために重要です。 1。デジタルセンサーアレイ LUCASプラットフォームは、デジタル、個々のセルのホログラムを記録する光センサアレイを利用しています。使用することができます：または相補型金属酸化膜半導体チップ（MT9P031、マイクロンCMOS、サンプルモデル）：、この目的のために、いくつかのデバイスを（KAI - 11002、KAF - 39000、コダックからのサンプルモデルCCD）にてご利用いただけます。それぞれ、アクティブな10平方センチメートルのFOV、18平方センチメートル、および24.4平方ミリメートルで、コダックのためのピクセルサイズはいくつかのデバイス、KAI - 11002、KAF - 39000を充電し、マイクロンCMOSイメージセンサーは9μm、6.8μmおよび2.2μmです。 [1-2]。 2。ライトソース他のほとんどの顕微鏡イメージングのモダリティとは異なり、LUCASは、レーザーを必要としないため、さらに簡単な発光ダイオード（LED）は照明用に使用することができます。波長可変光源を有効にするために、我々はまた、繊維の束（77564、ニューポート）とのピンホールで構成される標準グレードフューズドシリカ繊維と一緒にキセノンランプ（コーナーストーンT260、ニューポート社）とモノクロメータを利用することができます〜直径100μm、センサ表面上に5〜10 cmの位置。この波長可変光源の構成は、細胞のホログラフィック署名が調整できる柔軟なプラットフォームを提供し、ハイブリッドデジタル署名は、優れた特性評価の精度と特異性のために信号対雑音比を向上させるために合成することができる。 [3] B.サンプルの調製とイメージングイメージングチップに基づいて、LUCASの概念実証は、下記のように不均一溶液を用いて実証される。同様のプロトコルは、様々な他の細胞タイプ[1-3]に適用することができる。 1。全血の希釈と不均一溶液の準備<ol><li>プロセスを開始するには、全血サンプルと様々な直径（5、10、20μmである。デュークサイエンティフィック）のポリスチレンビーズを取得し、室温にそれらのすべてをもたらす。 </li><li>滅菌5mLのポリプロピレンチューブに、希釈剤としてRPMI 1640古典的な液体培地（フィッシャーサイエンティフィック社）の2 mlを加え。 </li><li> 30分間の全血サンプルをインキュベートした後、沈降した赤血球試料の10μLをピペットおよびRPMI溶液中にそれを転送する。 </li><li>赤血球RPMIの希釈に20μLポリスチレンのマイクロビーズの合計量を追加し、穏やかにピペッティングしたり、攪拌棒でマグネチックスターラーを使用して、細胞溶液を攪拌。 </li><li>カバースリップの上に5から15μL不均一溶液の総量を配置し、鉗子を使用して、ソリューション秒以上、同一のカバースリップを配置。サンプルは均等に2つのカバースリップの間に挟まれるべきである。 </li><li>その後、真空ペン（NT57 - 636、エドモンドオプティクス）を使用すると、イメージングのためのセンサアレイの活性領域にサンプルをロードします。 </li></ol> 2。全血の染色<ol><li> 0.1％の混合物の製造から始めては電力型の純粋なエオシンY（MW = 691.85、アクロスオーガニック）、亜鉛を含​​まない純粋な新規メチレンブルー色素（MW = 347.90、アクロスオーガニック）を使用して、エオシンYおよび希薄化後のニューメチレンブルー溶液をバッファとシュウ酸カリウム一水和物（99.0％試薬ACS、アクロスオーガニック）、各溶液は、水性白血球の染色のための0.45μmの孔径Syringlessフィルター（ワットマン）で精製される。 </li><li>その後、静かに2分間マグネチックスターラーを使用することによりポリプロピレンのビーカーで1:1の体積比で全血検体を溶解した水溶液染色試薬を攪拌し、10分間インキュベートする。 </li><li> 5mLのポリプロピレンチューブに50μLのステンド血液を移し、1:200の体積比を取得するために1.95 mLのRPMI 1640古典的な溶液（フィッシャーサイエンティフィック）で希釈する。その後、渦30秒のための希釈。異なる希釈率も、この段階で導入することができる。 </li><li>カバーガラスの間やマイクロ流体貯留層内で10〜100μLの染色細胞液をピペットで。その後、真空ペン（NT57 - 636、エドモンドオプティクス）を使用して、センサアレイの活性領域の上に試料を配置。 </li></ol>駐在結果ve_content&quot;&gt; <img src="/files/ftp_upload/1650/1650Fig1tmb.jpg" alt="図1" border="0" /> 図1：LUCASはどんなレンズ、レーザーや他の大型の光学部品を使用せずに正確なカウントと様々な細胞とその2次元ホログラフィック署名に基づくマイクロオブジェクトのデジタル分化を可能にする。様々なマイクロオブジェクトの特性LUCAS署名は、この図に示されていると40倍対物レンズで得られた従来の顕微鏡画像と比較されます。してください<a href="https://www.jove.com/files/ftp_upload/1650/1650Fig1.jpg">ここをクリックして</a>図1の拡大バージョンを参照すること。 <img src="/files/ftp_upload/1650/1650Fig2.jpg" alt="図2" /> 図2：赤血球、10um、5um、3 UM粒子を含む不均一な混合物の（左）RAW画像。ビューの同じフィールド（右）全自動LUCASの特性評価の結果が示されている。意思決定のアルゴリズムは3umビーズのような雑音比が低い信号で高密度領域だけでなく、粒子の特性に堅牢であることに注意してください。 <img src="/files/ftp_upload/1650/1650Fig3tmb.jpg" alt="図3" border="0" /> 図3：LUCASカスタムのインターフェースが示されている。 JavaベースのLUCASソフトウェアは、センサの画素サイズや光の波長など、さまざまな実験条件の入力が可能になります。 image上のビューの特定のフィールドの選択を行うこともできるし、標的細胞のパターンは統計的な細胞の影のライブラリを構築するためにユーザが定義することができます。取得したルーカスのイメージは、この訓練データに基づいて特徴づけることができる（すなわち、セルの影のライブラリ）とマーク（カウント）のイメージがユーザーに表示されます。カウント結果の統計情報も、さらなる分析のためのXMLファイルとして保存されます。

<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>

我々は、LUCASプラットフォームが正確にカウントし、そのホログラフィック署名に基づくチップ上に様々なmicro-objects/cellsを識別できるよう図示、およびポイントオブケア医療診断およびハイスループット細胞生物学のための有望なツールを提供している。正確に記録されたホログラムパターンを処理するために、我々は独自に開発したLUCASの意思決定のソフトウェアを実装。ルーカスの画像?...

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<td>Charged couple device (CCD)</td>
<td>&nbsp;</td>
<td>KODAK</td>
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<td>&nbsp;</td>
<td>KODAK</td>
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<td>&nbsp;</td>
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<td>Complementary Metal-Oxide-Semiconductor (CMOS)</td>
<td>&nbsp;</td>
<td>Micron</td>
<td>MT9P031</td>
<td>&nbsp;</td>
<tr>
<td>Xenon Lamp</td>
<td>&nbsp;</td>
<td>Newport Corp.</td>
<td>Cornerstone T260</td>
<td>&nbsp;</td>
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<td>Vacuum pen</td>
<td>&nbsp;</td>
<td>Edmund Optics</td>
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<td>4000 Series</td>
<td>&nbsp;</td>
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<td>RPMI</td>
<td>&nbsp;</td>
<td>Fisher Scientific</td>
<td>1640</td>
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<td>Pure Eosin Y</td>
<td>&nbsp;</td>
<td>Acros Organics</td>
<td>MW=691.85</td>
<td>&nbsp;</td>
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<td>New Methylene Blue(NMB) Dye</td>
<td>&nbsp;</td>
<td>Acros Organics</td>
<td>MW=347.90</td>
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<td>Potassium Oxalate Monohydrate</td>
<td>&nbsp;</td>
<td>Acros Organics</td>
<td>99.0% Reagent ACS</td>
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lensless on-chip imaging of cells provides a new tool for high-throughput cell-biology and medical diagnostics

他のレンズと光学部品と一緒に動作する対物レンズの使用により、従来の光学顕微鏡画像の細胞。非常に効果的ですが、この古典的なアプローチは、マイクロフルイディクスの技術の高度な状態と互換性を持たせるためにイメージングプラットフォームの小型化に一定の制限があります。このレポートでは、我々はLUCASと呼ばlenslessオンチップイメージングコンセプトの実験の詳細を紹介する（<strong<em&gt; L</em</strong&gt; ensless<strong<em&gt; U</em</strong&gt; LTRAワイド視野<strong<em&gt; C</em</strong&gt;エルのモニタリング<strong<em&gt;</em</strongに基づいて&gt; rrayプラットフォーム<strong<em&gt; S</em</strong同じ大きさにまたがることができるビューの超広視野上の画像不均一な細胞のソリューションを、任意の顕微鏡対物レンズや他の大型の光学部品を必要としない&gt;ハドウイメージング）〜18センチメートル<sup&gt; 2</sup&gt;。また、従来の顕微鏡とは違って、LUCAS〜9 mLのサンプル容量を最大に対応するリフォーカスすることなく画像〜5 mmの被写界深度の上の関心の異種細胞溶液をすることができます。このイメージングプラットフォームレコードその視野内で関心の各セルの影（すなわち、lenslessデジタルホログラム）、およびこれらの細胞の影の自動化されたデジタル処理は、タイプ、カウントおよびソリューション内のセルの相対位置を決定することができます。それはどんなかさばる光学部品または機械的なスキャニング段階が必要としないため、同時にcare診断ツールの特にポイントのために非常に重要であるコストを、削減さを大幅に小型化されたプラットフォームを提供しています。さらに、このプラットフォームのイメージングのスループットは、ハイスループット細胞生物学実験のために極めて貴重であることが、従来の光学顕微鏡よりも優れて桁違いです。

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

細胞のLensfreeオンチップのイメージングと特性が示されている。このオンチップ細胞イメージングのアプローチは、リソースの乏しい環境のために特に適し、医療診断およびハイスループット細胞生物学のアプリケーション向けのコンパクトで費用効果の高いツールを提供します。

細胞のLenslessオンチップイメージングは​​、ハイスループット細胞生物学および医学診断のための新しいツールを提供します。

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 ビュー.  University of California, Los Angeles. 細胞のLensfreeオンチップのイメージングと特性が示されている。このオンチップ細胞イメージングのアプローチは、リソースの乏しい環境のために特に適し、医療診断およびハイスループット細胞生物学のアプリケーション向けのコンパクトで費用効果の高いツールを提供します。

ビデオ: 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.

気管切開：肺へ幹細胞の移植方法

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 ...

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.

細胞生物学の勾配を生むマイクロ流体デバイス

 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.

内脂肪量の生化学的および高スループット微視的評価<em&gt;線虫（Caenorhabditis elegans）</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.

抗核抗体のHE​​p-2細胞を用いたスクリーニング

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.

腫瘍球の高スループット画像解析：自動的かつ正確にスフェロイドのサイズを測定するための使いやすいソフトウェアアプリケーション

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.

転写後調節される遺伝子の化学モジュレーターのためのハイスループットスクリーニング

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.

微細藻類のモデル改良のための高スループット代謝プロファイリング

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.

成長と遺伝子発現の不均一性を測定するマイクロコロニーのハイスループットライブイメージング

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.

細胞DNA損傷を評価するためのハイスループットコメットアッセイアプローチ

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.

オルガノイドの培養および3Dイメージングのためのハイスループットプラットフォーム

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

細胞のLenslessオンチップイメージングは​​、ハイスループット細胞生物学および医学診断のための新しいツールを提供します。

細胞のLenslessオンチップイメージングは、ハイスループット細胞生物学および医学診断のための新しいツールを提供します。