<html> Burada LUCAS [1-3] dahil olan deneysel prosedürleri tartışacağız. LUCAS kavramı kanıtı göstermek için biz bütün bir kan örneği görüntüleme işlemi anlatacağım. A. Görüntüleme Set-up LUCAS Görüntüleme platformu, özellikle kaynak sınırlı ayarları için bir maliyet-etkin ve kompakt bir alternatif, mevcut bakım noktası sitometri ve tıbbi tanı araçları sağlamak için önemli avantajlar sergiliyor. LUCAS hücrelerin görüntü tespit yerine, her bir hücrenin arka plan ışığı ile dağınık ışık girişim tarafından oluşturulan hücrelerin yerine dijital hologramlar yakalar. Aydınlatma kısmi mekansal tutarlılığı dikkatli kontrol holografik kayıt etkinleştirmek için kritik öneme sahiptir. 1. Dijital sensör dizisi LUCAS platformu optoelektronik sensör, dijital olarak tek tek hücre hologramlar kaydetmek için bir dizi kullanır. Kullanılabilir: veya tamamlayıcı metal oksit yarı iletken çip (MT9P031, Micron CMOS, Örnek Model): Bu amaçla, çift cihazlar (KAI-11002, Kodak KAF-39000, Örnek Modeller CCD) ücret. Sırasıyla, aktif bir 10 cm2 FOV, 18 cm2, ve 24.4 mm2 Kodak piksel boyutlarında çift cihazları, KAI-11002, KAF-39000 ücret ve Micron CMOS görüntü sensörleri, 9 mm, 6.8 mm ve 2.2 mm. [1-2]. 2. Işık kaynağı Diğer çoğu mikroskopik görüntüleme yöntemlerinin aksine, LUCAS, bu nedenle bir lazer ve hatta basit bir ışık yayan diyot (LED) aydınlatma için kullanılan gerektirmez. Dalga boyu ayarlanabilir aydınlatma sağlamak için, biz de liflerin paket (77.564, Newport) ve iğne deliği oluşan standart bir not erimiş silika elyaf ile birlikte bir Xenon Lamba (Cornerstone T260, Newport Corp) ile monokromatör yararlanabilirler ~~ 100 mikron çapında ~ sensör yüzeyi üzerinde 5-10 cm bulunmaktadır. Bu ayarlanabilir dalga boyu aydınlatma yapılandırma hücrelerin holografik imzaları ayarlanabilir esnek bir platform sağlar ve melez dijital imzalar sinyal gürültü oranı daha iyi karakterizasyonu doğruluk ve özgüllük için geliştirmek için sentezlenmiş olabilir. [3] B. Numune Hazırlama ve Görüntüleme Aşağıda açıklandığı gibi heterojen bir çözüm kullanarak görüntüleme-çip tabanlı LUCAS kavramı bir kanıtı sunulacaktır. Benzer bir protokol, çeşitli hücre tipleri [1-3] için uygulanabilir. 1. Tam kan sulandırma ve heterojen bir çözümün hazırlanması <ol><li> Sürecine başlamak için, bütün bir kan örneği ve çeşitli çaplarda (5, 10 ve 20 mikron. Duke Bilimsel) polistiren mikro almak ve hepsi oda sıcaklığına getirmek. </li><li> RPMI 1640 classic sıvı ortam (Fisher Scientific) bir seyreltici olarak steril bir 5 ml polipropilen tüpe 2 mL ekleyin. </li><li> 30 dakika boyunca tam kan inkübe sonra, 10 mcL tortulaşmış eritrosit örnek pipet ve RPMI çözüm içine aktarın. </li><li> 20 mcL polistiren mikro toplam hacmi, eritrosit RPMI seyreltme içine ekleyin ve hafifçe pipetleme veya bir heyecan çubuğu ile manyetik karıştırıcı kullanılarak hücre çözümü kışkırtmak. </li><li> 5-15 mcL heterojen bir çözüm toplam hacmi, forseps kullanarak ikinci bir çözüm üzerinde, aynı kapak kayma kayma ve yerde bir kapak üzerine yerleştirin. Örnek eşit iki kapak fişleri arasında sıkışmış olmalı. </li><li> Sonra, bir vakum kalem (NT57-636, Edmund Optik) kullanarak görüntüleme sensörü dizinin aktif bölge üzerine örnek yüklemek. </li></ol> 2. Tam Kan Boyama <ol><li> % 0.1 &#39;lik bir karışım hazırlanması ile başlayan güç tipi, saf Eosin Y (MW = 691,85, Acros Organics), çinko içermeyen saf Yeni Metilen Mavi boya (MW = 347,90, Acros Organics) kullanarak Eosin Y ve seyreltilmiş Yeni Metilen Çözüm tamponlu Potasyum Oksalat Monohidrat (99.0% Reaktif ACS, Acros Organik); her çözüm 0,45 mikron gözenek boyutu Syringless Filtresi (Whatman) sulu beyaz kan hücre boyama ile arındırılır. </li><li> Sonra, yavaşça, 2 dakika boyunca bir manyetik karıştırıcı kullanılarak bir hacim oranı 1:1 bir polipropilen beher tam kan örneği ile çözünmüş sulu boyama reaktif karıştırın ve 10 dakika inkübe edin. </li><li> 5 ml polipropilen tüpe 50 mcL lekeli bir kan çözüm aktarın ve 1:200 bir hacim oranı elde etmek için RPMI 1640 classic 1.95 ml sıvı solüsyon (Fisher Scientific) ile seyreltilir. Daha sonra, 30 saniye için seyreltme girdap. Farklı bir seyreltme oranı da bu aşamada devreye alınabilir. </li><li> Kapak fişleri arasında veya bir mikroakışkan rezervuar içinde 10-100 mcL lekeli bir hücre çözümü Pipet. Sonra, bir vakum kalem kullanarak (NT57-636, Edmund Optik) numune, sensör dizisi aktif bölge üzerine yerleştirin. </li></ol> TEMSİLCİSİ SONUÇLAR ve_content &quot;&gt; <img src="/files/ftp_upload/1650/1650Fig1tmb.jpg" alt="Şekil 1" border="0" /> Şekil 1: LUCAS doğru sayma ve çeşitli hücreler ve mikro-nesneleri lensler, lazerler veya diğer hantal optik bileşenleri kullanarak 2 boyutlu holografik imzalar dayalı dijital farklılaşma sağlar. Karakteristik LUCAS imzaları çeşitli mikro-nesneleri bu resimde gösterildiği ve 40X objektif lens ile elde edilen geleneksel bir mikroskop görüntü karşılaştırılır. Lütfen Şekil 1&#39;de bir büyük halini görmek için <a href="https://www.jove.com/files/ftp_upload/1650/1650Fig1.jpg">buraya tıklayınız</a> . <img src="/files/ftp_upload/1650/1650Fig2.jpg" alt="Şekil 2" /> Şekil 2: (Sol) Ham görüntü, kırmızı kan hücreleri, 10um, 5um ve 3 um parçacıklar içeren heterojen bir karışım . Aynı alan için (Sağ) Tam otomatik LUCAS karakterizasyonu sonuçları gösterilmiştir. Karar algoritması 3um boncuklar gibi gürültü oranı yüksek yoğunluklu bölgelerinde, düşük sinyal ile parçacıkların yanı sıra karakterize sağlam olduğunu unutmayın. <img src="/files/ftp_upload/1650/1650Fig3tmb.jpg" alt="Şekil 3" border="0" /> Şekil 3: LUCAS özel arayüz gösterilmiştir. Java tabanlı LUCAS yazılımı gibi sensör piksel boyutu veya ışık dalga boyu gibi çeşitli deneysel koşullar için girdi sağlar. Resmin üzerine görüş belirli bir alan seçimi de yapılabilir ve hedef hücre modelleri, istatistiksel bir hücre gölge kütüphane oluşturmak için kullanıcı tarafından tanımlanabilir. , Elde edilen LUCAS görüntü, daha sonra bu eğitim verileri (örneğin, hücre gölge kütüphane) ve işaretlenmiş (sayılır) resim kullanıcıya görüntülenen dayalı karakterize edilebilir. Sayımı sonuçları, istatistikler, aynı zamanda daha fazla analiz için bir XML dosyası olarak depolanır.

<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>

LUCAS platformu doğru saymak ve holografik imzaları dayalı bir çip üzerinde çeşitli micro-objects/cells tespit gösterildiği gibi, noktası bakım, tıbbi teşhis ve yüksek verimli hücre biyolojisi için umut vaat eden bir araç sağlar. Doğru kaydedilen holografik desen işlemek için geliştirilmiş özel bir LUCAS karar yazılım uygulanmaktadır. LUCAS görüntülerin eğitim tarafından oluşturulan bir istatistik kırınım görüntü veritabanını kullanır Bu algoritma, heterojen bir çözüm içinde...

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		<table border="1">
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		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>Charged couple device (CCD)</td>
<td>&nbsp;</td>
<td>KODAK</td>
<td>KAI-11002</td>
<td>&nbsp;</td>
<tr>
<td>Charged couple device (CCD)</td>
<td>&nbsp;</td>
<td>KODAK</td>
<td>KAF-39000</td>
<td>&nbsp;</td>
<tr>
<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>
<tr>
<td>Vacuum pen</td>
<td>&nbsp;</td>
<td>Edmund Optics</td>
<td>NT57-636</td>
<td>&nbsp;</td>
<tr>
<td>5, 10, and 20 &mu;m Microbeads</td>
<td>&nbsp;</td>
<td>Thermo Scientific</td>
<td>4000 Series</td>
<td>&nbsp;</td>
<tr>
<td>RPMI</td>
<td>&nbsp;</td>
<td>Fisher Scientific</td>
<td>1640</td>
<td>&nbsp;</td>
<tr>
<td>Pure Eosin Y</td>
<td>&nbsp;</td>
<td>Acros Organics</td>
<td>MW=691.85</td>
<td>&nbsp;</td>
<tr>
<td>New Methylene Blue(NMB) Dye</td>
<td>&nbsp;</td>
<td>Acros Organics</td>
<td>MW=347.90</td>
<td>&nbsp;</td>
<tr>
<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

Diğer lens ve optik bileşenlerin birlikte çalışan nesnel lenslerin kullanımı ile Konvansiyonel optik mikroskoplar görüntü hücreleri. Oldukça etkili olmakla birlikte, bu klasik yaklaşım Mikroakiskan sanat gelişmiş devlet ile uyumlu hale getirmek için görüntüleme platformu minyatürleştirme için bazı sınırlamalar vardır. Bu raporda, LUCAS olarak adlandırılan, bir lensless çip üzerinde görüntüleme kavramının deneysel detaylar (tanıtmak<strong<em&gt; L</em</strong&gt; Ensless<strong<em&gt; U</em</strong&gt; LTRA geniş alan görüş<strong<em&gt; C</em</strong&gt; Ell izleme<strong<em&gt; A</em</strong&gt; Rray platformu dayalı<strong<em&gt; S</em</strong, Görüntü gibi büyük açıklıklı bir ultra geniş alan heterojen bir hücre çözümü hedefleri için herhangi bir mikroskop veya diğer hantal optik bileşenleri gerektirmez&gt; hadow görüntüleme) ~ 18 cm<sup&gt; 2</sup&gt;. Ayrıca, geleneksel mikroskoplar aksine, LUCAS görüntü, ~ 9 ml numune hacmi karşılık refocusing gerek kalmadan ~ 5 mm derinlik-alan üzerinde ilgi heterojen bir hücre çözümü. Bu görüntüleme platformu, tipi, sayısı ve çözüm içindeki hücrelerin göreli konumları belirlemek, her bir hücre, görüş alanı içinde ilgi ve bu hücre gölgelerin otomatik dijital işleme (yani, lensless dijital hologramlar) gölgesinde kaydeder. Hantal herhangi bir optik bileşenleri veya mekanik tarama aşamaları gerektirmez, çünkü, aynı zamanda, bakım tanılama araçları özellikle noktası için oldukça önemlidir maliyeti, azalttığını önemli ölçüde minyatür bir platform sunmaktadır. Ayrıca, bu platformun görüntüleme hacmi yüksek verimli hücre biyoloji deneyleri için son derece değerli olabilir geleneksel optik mikroskoplar, daha iyi büyüklükte emir.

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

Hücre Lensfree çip üzerinde görüntüleme ve karakterizasyonu gösterilmiştir. Bu çip üzerinde hücre görüntüleme yaklaşımı, kaynak yoksul ayarları için özellikle uygun hale, tıbbi teşhis ve yüksek verimli hücre biyolojisi uygulamaları için kompakt ve maliyet-etkin bir araç sağlar.

Hücre Lensless-çip Görüntüleme Yüksek throughput Hücre Biyolojisi ve Tıbbi Teşhis için Yeni Aracı sağlar.

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 Görüntüleme.  University of California, Los Angeles. Hücre Lensfree çip üzerinde görüntüleme ve karakterizasyonu gösterilmiştir. Bu çip üzerinde hücre görüntüleme yaklaşımı, kaynak yoksul ayarları için özellikle uygun hale, tıbbi teşhis ve yüksek verimli hücre biyolojisi uygulamaları için kompakt ve maliyet-etkin bir araç sağlar.

Video: 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.

Trakeotomi: Akciğer Kök Hücre Nakli Bir Yöntem

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 ...

Biyoloji

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.

Hücre Biyolojisi Degrade üreten mikroakışkan Aygıt

 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.

Fat Kütle Biyokimyasal ve Yüksek Verimli Mikroskobik Değerlendirme<em&gt; 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.

Anti-Nükleer Antikor HEp-2 hücreleri kullanarak Eleme

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.

Tümör sferoidlerin Yüksek throughput Görüntü Analizi: Otomatik ve Doğru küremsiler boyutunu ölçmek için kullanıcı-dostu Yazılım Uygulama

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.

Post-transkripsiyonel ayarlı genlerin kimyasal Modülatörler için yüksek verimli tarama

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.

Mikroalglerin Model İyileştirmeleri için Yüksek Verimli Metabolik Profil oluşturma

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.

Büyüme ve Gen İfadesinde Heterojenliği Ölçmek için Mikrokolonların Yüksek Verimli Canlı Görüntülemesi

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.

Hücresel DNA Hasarını Değerlendirmek için Yüksek Verimli Bir Kuyruklu Yıldız Testi Yaklaşımı

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.

Organoidlerin kültürü ve 3D görüntülenmesi için yüksek verimli bir platform

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