Bu çalışma Hukriede, NCI / NIH (P01 CA78039) Vogt ve NHLBI / NIH (1R01HL088016) Tsang için NICHD / NIH (1R01HD053287) tarafından finanse edilmektedir.

1) Zebra balığı çiftleşmelerin ve BCI tedavi ayarlayın. <ol><li> Bireysel çiftleşme tankları 1&#39;e pt6 Zebra balığı ve yeri: İki gün önce tedavi, homozigot erkek Tg (d2eGFP dusp6) tanımlamak. Ayırıcı ve vahşi bir türü AB * dişi balık ekleyin ve çiftleşme kafesleri bir gecede terk. </li><li> Ertesi sabah ayırıcı kaldırmak ve 1 saat sonra embriyolar toplamak. E3 (5 mM NaCl, 0.17 mM KCl, 0.33 mM CaCl2, 0.33 mM MgSO4), çözüm ve 6 saat boyunca 28 ° C&#39;de inkübe 100mm yemekleri embriyo transferi. Ölü kaldırın ve gübresiz embriyolar, taze E3 bir çözüm ekleyin ve bir gece inkübe edin. </li><li> Benzer evre (24hpf) ve uniform GFP ifade transgenik embriyolar sıralayın. 96 plaka her kuyuya ayrı ayrı Dizi embriyolar. </li><li> Mikropipet aşırı E3 çözüm çıkarın. E3 çözüm her kuyuya bir çok kanallı pipet ile 200μl ekleyin. </li><li> Olarak listelenen aşağıdaki bileşikler ekle: <table border="1"><tbody><tr><td></td><td> 1 </td><td> 2 </td><td> 3 </td><td> 4 </td><td> 5 </td><td> 6 </td><td> 7 </td><td> 8 </td><td> 9 </td><td> 10 </td><td> 11 </td><td> 12 </td></tr><tr ><td> A </td><td> 2μl DMSO </td><td> 2μl BCL (0.1mm) </td><td> 2μl BCL (0.5mm) </td><td> 2μl BCL (1mm) </td><td> 2μl BCL (2mm) </td><td> 2μl BCL (2.5mm) </td><td> 2μl BCL (0.1mm) </td><td> 2μl BCL (0.5mm) </td><td> 2μl BCL (1mm) </td><td> 2μl BCL (2mm) </td><td> 2μl BCL (2.5mm) </td><td> 2μl DMSO </td></tr><tr ><td> B </td><td> 2μl DMSO </td><td> 2μl BCL (0.1mm) </td><td> 2μl BCL (0.5mm) </td><td> 2μl BCL (1mm) </td><td> 2μl BCL (2mm) </td><td> 2μl BCL (2.5mm) </td><td> 2μl BCL (0.1mm) </td><td> 2μl BCL (0.5mm) </td><td> 2μl BCL (1mm) </td><td> 2μl BCL (2mm) </td><td> 2μl BCL (2.5mm) </td><td> 2μl DMSO </td></tr><tr ><td> C </td><td> 2μl DMSO </td><td> 2μl BCL (0.1mm) </td><td> 2μl BCL (0.5mm) </td><td> 2μl BCL (1mm) </td><td> 2μl BCL (2mm) </td><td> 2μl BCL (2.5mm) </td><td> 2μl BCL (0.1mm) </td><td> 2μl BCL (0.5mm) </td><td> 2μl BCL (1mm) </td><td> 2μl BCL (2mm) </td><td> 2μl BCL (2.5mm) </td><td> 2μl DMSO </td></tr><tr ><td> D </td><td> 2μl DMSO </td><td> 2μl BCL (0.1mm) </td><td> 2μl BCL (0.5mm) </td><td> 2μl BCL (1mm) </td><td> 2μl BCL (2mm) </td><td> 2μl BCL (2.5mm) </td><td> 2μl BCL (0.1mm) </td><td> 2μl BCL (0.5mm) </td><td> 2μl BCL (1mm) </td><td> 2μl BCL (2mm) </td><td> 2μl BCL (2.5mm) </td><td> 2μl DMSO </td></tr><tr ><td> E </td><td> 2μl DMSO </td><td> 2μl BCL (0.1mm) </td><td> 2μl BCL (0.5mm) </td><td> 2μl BCL (1mm) </td><td> 2μl BCL (2mm) </td><td> 2μl BCL (2.5mm) </td><td> 2μl BCL (0.1mm) </td><td> 2μl BCL (0.5mm) </td><td> 2μl BCL (1mm) </td><td> 2μl BCL (2mm) </td><td> 2μl BCL (2.5mm) </td><td> 2μl DMSO </td></tr><tr ><td> F </td><td> 2μl DMSO </td><td> 2μl BCL (0.1mm) </td><td> 2μl BCL (0.5mm) </td><td> 2μl BCL (1mm) </td><td> 2μl BCL (2mm) </td><td> 2μl BCL (2.5mm) </td><td> 2μl BCL (0.1mm) </td><td> 2μl BCL (0.5mm) </td><td> 2μl BCL (1mm) </td><td> 2μl BCL (2mm) </td><td> 2μl BCL (2.5mm) </td><td> 2μl DMSO </td></tr><tr ><td> G </td><td> 2μl DMSO </td><td> 2μl BCL (0.1mm) </td><td> 2μl BCL (0.5mm) </td><td> 2μl BCL (1mm) </td><td> 2μl BCL (2mm) </td><td> 2μl BCL (2.5mm) </td><td> 2μl BCL (0.1mm) </td><td> 2μl BCL (0.5mm) </td><td> 2μl BCL (1mm) </td><td> 2μl BCL (2mm) </td><td> 2μl BCL (2,5 mM) </td><td> 2μl DMSO </td></tr><tr ><td> H </td><td> 2μl DMSO </td><td> 2μl BCL (0.1mm) </td><td> 2μl BCL (0.5mm) </td><td> 2μl BCL (1mm) </td><td> 2μl BCL (2mm) </td><td> 2μl BCL (2.5mm) </td><td> 2μl BCL (0.1mm) </td><td> 2μl BCL (0.5mm) </td><td> 2μl BCL (1mm) </td><td> 2μl BCL (2mm) </td><td> 2μl BCL (2.5mm) </td><td> 2μl DMSO </td></tr></tbody></table> Final konsantrasyonları, 1% DMSO 5μM, 10μM, 20μM, 25μM BCI 1 mikrona kadar. Kapak ve alüminyum folyo levha az 6 saat boyunca inkübe 28 ° C </li></ol> 2) 96-kuyucuğu ve analiz Otomatik görüntüleme. <ol><li> 30hpf embriyolar uyutmak için her iyi tricaine (0.61mM) 10μl ekleyin. </li><li> Moleküler Cihazlar Imagexpress Ultra içine plaka yükleyin. </li><li> Açık Metaxpress yazılım. 4x amacı ve maksimum iğne deliği çapı ile bir yapılandırma seçin, lazer otofokus plaka alt algılamak için yapılandırmak ve Z-düzleminde ilgi bölgeleri tespit etmek için en iyi belirlemek. 488 / 525 nm (GFP) uyarma / emisyon dalga boylarında görüntüler elde. Hiçbir binning ile 2000x2000 piksel (4 x 4 mm), tek bir site olarak alan tarama ayarlayın. Lazer güç yapılandırma ve pozitif kontrol görüntüler gibi parlak bölgelerde elde doyurabilecek değil. Birçok pozitif ve negatif kontrol kuyuları düzgün bir görüntü elde etme onaylayın. </li><li> Plaka tarama başlatın. Cihaz, otomatik olarak tüm kuyuları ve arşiv onları bir SQL Server veritabanı görüntüleri kazanacaklardır. </li><li> Definiens Geliştirici Cellenger uygulama modülü ve Moleküler Aygıtlar Imagexpress Ultra platformu tarafından oluşturulan görüntü formatı ve dosya yapısını tanıyan bir ithalat filtre kullanarak resim gönderin. </li><li> Özel olarak tasarlanmış bir Biliş Ağ Teknolojisi algoritma (aynı zamanda bir kural setleri olarak adlandırılır) ile işlem görüntüler. Biz özel kısa bir süre önce yayınlanan kuralkümesi 2 dayalı geliştirilen algoritma, embriyo, yumurta sarısı, ve baş otomatik olarak tespit etmek ve kafa yapıları GFP-ifade parlaklık ve alanını ölçmek için tasarlanmıştır . Embriyo tespit edildiği veya kafası net bir şekilde atanan (örneğin, odak görüntüleri yüksek bileşik toksisite veya dışarı) olamaz nerede Wells analiz dışında tutulmuştur. Algoritma kullanarak birkaç negatif kontrol görüntüler (araç tedavi) ve pozitif kontrol görüntüler (BCI tedavi) baş ve yumurta sarısı, hem de doğru tanımlanmış olması gibi yapılandırın. Parlak kafa yapılarının tespiti görsel gözlem eşleşen kadar ortalama sarısı yoğunluğu katları olarak bir &quot;kafa yapıları&quot; algılama eşiği ayarlayın. Ihracat parametreleri tanımlayın. Bu örnekte, en bilgilendirici parametre bundan sonra &quot;toplam kafa yapıları parlaklık&quot; (Şekil 1) olarak adlandırılan başın içinde GFP pozitif bölgelerde, toplam entegre parlaklık oldu. <img src="/files/ftp_upload/1900/1900fig2.jpg" alt="Şekil 1" /> Şekil 1 Grafik transgenik embriyolarda GFP ifade BCI doza bağımlı etkileri depicitng. </li><li> Algoritma kullanan tüm entegre iş scheduler plakası görüntüleri çalıştırın. Definiens yazılım aynı anda birden fazla görüntü analizi sağlayan dağıtılmış bilgi işlem, birkaç işlemci (&quot;motorları&quot;) kullanır. Algoritması uygulanan analizi ile işlenmiş görüntünün bir kopyası ile birlikte veritabanına kaydedilir görüntü analizi, elde edilen sonuçlar dayalı sayısal ölçümler hesaplar. </li></ol> Temsilcisi Sonuçlar: Sonra tüm görüntüleri analiz, veri Cellenger içinde görüntülenir ya da daha fazla analiz ve grafiksel gösterimi için üçüncü parti yazılımlar (Excel, GraphPad Prism) ihraç. Şekil 2 tedavi edilen bir araç tarama görüntüler arşivlenir ve iyi uygulanmış ve CNT analiz olmadan BCI-tedavi bir. Şekil 1 belgeleri Tg GFP muhabiri gen ekspresyonu (dusp6: d2EGFP) BCI, doza bağımlı bir etkisi pt6 embriyolar. Yarım maksimum yanıt (EC50) ortaya çıkarmak için gerekli konsantrasyonu yaklaşık 12 mcM idi. <img src="/files/ftp_upload/1900/1900fig1.jpg" alt="Şekil 2" /> Şekil 2 Transgenik zebrafish embriyoların Otomatik görüntü yakalama ve analizi. 30hpf pt6 embriyo: (A) DMSO araç Tg (d2eGFP dusp6) tedavi. (B) 20μM BCI gösterisi ile tedavi edilen transgenik embriyolar GFP ifade artırır. (C) embriyo Görüntü (A) baş GFP ifade bulmak için kural setleri Definiens çalıştırdıktan sonra. Turuncu noktalar yazılım GFP yoğunluğunu ölçen göstermektedir. (D) embriyo Görüntü, (B) baş GFP ifade bulmak için kural setleri Definiens çalıştırdıktan sonra. Turuncu noktalar yazılım GFP yoğunluğunu ölçen göstermektedir. Kuralkümesi tedavi edilen embriyo GFP ifade genişletilmiş bir alan bulmak mümkün olduğunu unutmayın.

<ol>
	<li>Molina, G. A., Watkins, S. C., Tsang, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Generation+of+FGF+reporter+transgenic+zebrafish+and+their+utility+in+chemical+screens.">Generation of FGF reporter transgenic zebrafish and their utility in chemical screens.</a> BMC Dev Biol. 7, 62-62 (2007).</li><li>Vogt, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Automated+image-based+phenotypic+analysis+in+zebrafish+embryos.">Automated image-based phenotypic analysis in zebrafish embryos.</a> Dev Dyn. 238, 656-663 (2009).</li><li>Molina, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Zebrafish+chemical+screening+reveals+an+inhibitor+of+Dusp6+that+expands+cardiac+cell+lineages.">Zebrafish chemical screening reveals an inhibitor of Dusp6 that expands cardiac cell lineages.</a> Nat Chem Biol. 5, 680-687 (2009).</li></ol>

Zebrafish kimyasal ekranların verimini sınırlayan en önemli faktör, sistematik bir süreç görüntüleme ve analiz için 96-kuyucuğu metodoloji eksikliği. Çok hücreli organizmalarda görüntüleri karmaşık, düşük kontrast ve heterojen doğası nedeniyle, mevcut görüntü analizi algoritmaları organizma içinde özel yapıları tespit etmek ve ölçmek için başarısız olur. En yayımlanan zebrafish kimyasal ekranları bu nedenle işlem boyunca özverili personeli tarafından bireysel kuyuların gözle muayene içerir. Bu genellikle sayısal okuma üretim ve tam bir ekran görüntüleri ve veri arşivleme önler. Ayrıca, elle değerlendirme, yani görüntü tabanlı tarama, ekran performansı retrospektif analizler yapmak için tarama kampanyası (örneğin, toksisite ya da gelişimsel kusurlar) birincil odak fenotipik değişiklikleri incelemek ve çapraz yeteneği çok önemli avantajları ortadan kaldırır geçmiş veya gelecekteki ekranlar ile aynı bileşik kitaplığı kullanılarak referans tarama verileri. Bu yazıda, kullanıcının müdahalesine gerek kalmadan zebrafish embriyoların multiwell plakalar otomatik görüntüleme ve analiz için metodoloji vurgulayın. Biz 96 kuyucuğu pt6 embriyo ve Definiens Biliş Ağ Teknolojisi ki sayısal GFP dayalı bir görüntü analizi algoritması geliştirilen görüntü Tg (d2EGFP dusp6): konfokal okuyucu (Moleküler Aygıtlar, Sunnyvale, CA) tarama ImageXpress Ultra lazer otomatik görüntü yakalama transgenik embriyoların başkanları ifade. Yöntemi kademeli yanıtları teslim ve FGF sinyalizasyon küçük bir molekül aktivatör in vivo aktivite nicel. Zebrafish embriyoların 2, piyasada bulunan plaka okuyucuları çeşitli otomatik görüntü yakalama uygulamak mümkün olduğunu belgeleyen Epifloresans tabanlı ArrayScan II (Cellomics A.Ş., Pittsburgh, PA) ile benzer sonuçlar elde edildi . Çok hücreli organizma görüntüler otomatik olarak analiz etmek için metodoloji geliştirilmesi, insan bir gözlemci tarafından görsel skorlama için ihtiyaç ortadan nesil ve gelecek ekranları ile retrospektif analizi ve karşılaştırmalar için sayısal veri ve görüntü arşivleme etkin.

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<td>Tricaine (MS222)</td>
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development of automated imaging and analysis for zebrafish chemical screens.

Biz zebrafish embriyolar FGF / RAS / MAPK yolağı modüle küçük moleküller tanımlamak için görüntü tabanlı yüksek içerik tarama (HCS) metodolojisi uygulama göstermektedir. Zebra balığı embriyosu, in vivo yüksek içerikli kimyasal ekranlar için ideal bir sistem. 1 gün eski embriyo yaklaşık 1 mm çapında ve 96-kuyu plakalar, yüksek verim tarama için standart bir format içine kolayca dizilmiş olabilir. Gelişimin ilk gününde, embriyolar, böylece embriyogenez sırasında doku oluşumu görselleştirme sağlayan en önemli organların en şeffaf. Özellikle otomatik görüntü toplama ve analiz geliştirme zebrafish kimyasal ekranların tam otomasyon, ancak hala bir sorundur. Biz daha önce FGF aktivite kontrolü altında yeşil floresan proteininin (GFP) ifade eden transgenik muhabir hattı oluşturulmuş ve kimyasal ekranları kendi programını gösterdi<sup&gt; 1</sup&gt;. Yüksek verim tüm organizmanın ekranları için metodoloji kurmak için, biz 24-48 saat sonra döllenme (hpf) 96-kuyucuğu zebrafish embriyoların otomatik görüntüleme ve analiz için bir sistem geliştirdi<sup&gt; 2</sup&gt;. Bu video 24hpf multiwell tabak içine transgenik embriyolar arraying ilişkin usul vurgulayın ve küçük bir molekül (BCI) FGF sinyalizasyon hyperactivates ek<sup&gt; 3</sup&gt;. Plakalar tricaine Moleküler Aygıtlar ImageXpress Ultra lazer tarama konfokal HCS okuyucu otomatik görüntüleme önce larva uyutmak için ek takip 6 saat inkübe edilir. Görüntüler transgenik embriyoların başkanları GFP ifade algılamak ve ölçmek için geliştirilen bir Biliş Ağ Teknolojisi algoritması kullanılarak Definiens Developer yazılımı tarafından işlenir. Bu örnekte BCI tedavi edilen embriyolar GFP muhabiri gen ekspresyonu üzerine doza bağımlı etkileri ölçmek için algoritma yeteneği vurgulayın.

Watch this Scientific Journal Video about Development of automated imaging and analysis for zebrafish chemical screens. at JoVE.com

Development of automated imaging and analysis for zebrafish chemical screens.

Biz zebrafish transgenik embriyolar multiwell plakaları otomatik olarak görüntüleme ve analiz için bir sistem geliştirme raporu. Fibroblast Büyüme Faktörü muhabiri gen ekspresyonu üzerine küçük bir molekül, BCI, doza bağımlı etkileri ölçmek ve yüksek verimli zebrafish kimyasal ekranlar kurulması için teknoloji yeteneğini göstermektedir.

Zebrafish kimyasal ekranlar için otomatik görüntüleme ve analiz geliştirilmesi.

We demonstrate the application of image-based high-content screening (HCS) methodology to identify small molecules that can modulate the FGF/RAS/MAPK ...

development-automated-imaging-analysis-for-zebrafish-chemical

Research

JoVE Journal

Biology

11.3K Görüntüleme.  University of Pittsburgh Drug Discovery Institute. Biz zebrafish transgenik embriyolar multiwell plakaları otomatik olarak görüntüleme ve analiz için bir sistem geliştirme raporu. Fibroblast Büyüme Faktörü muhabiri gen ekspresyonu üzerine küçük bir molekül, BCI, doza bağımlı etkileri ölçmek ve yüksek verimli zebrafish kimyasal ekranlar kurulması için teknoloji yeteneğini göstermektedir.

Video: Development of automated imaging and analysis for zebrafish chemical screens.

A Neuronal and Astrocyte Co-Culture Assay for High Content Analysis of Neurotoxicity

High Content Analysis (HCA) assays combine cells and detection reagents with automated imaging and powerful image analysis algorithms, allowing measurement of multiple cellular phenotypes within a single assay. In this study, we utilized HCA to develop a novel assay for neurotoxicity. Neurotoxicity assessment represents an important part of drug safety evaluation, as well as being a significant focus of environmental protection efforts. Additionally, neurotoxicity is also a well-accepted in vitro marker of the development of neurodegenerative diseases such as Alzheimer's and Parkinson's diseases. Recently, the application of HCA to neuronal screening has been reported. By labeling neuronal cells with &beta;III-tubulin, HCA assays can provide high-throughput, non-subjective, quantitative measurements of parameters such as neuronal number, neurite count and neurite length, all of which can indicate neurotoxic effects. However, the role of astrocytes remains unexplored in these models. Astrocytes have an integral role in the maintenance of central nervous system (CNS) homeostasis, and are associated with both neuroprotection and neurodegradation when they are activated in response to toxic substances or disease states. GFAP is an intermediate filament protein expressed predominantly in the astrocytes of the CNS. Astrocytic activation (gliosis) leads to the upregulation of GFAP, commonly accompanied by astrocyte proliferation and hypertrophy. This process of reactive gliosis has been proposed as an early marker of damage to the nervous system. The traditional method for GFAP quantitation is by immunoassay. This approach is limited by an inability to provide information on cellular localization, morphology and cell number. We determined that HCA could be used to overcome these limitations and to simultaneously measure multiple features associated with gliosis - changes in GFAP expression, astrocyte hypertrophy, and astrocyte proliferation - within a single assay. In co-culture studies, astrocytes have been shown to protect neurons against several types of toxic insult and to critically influence neuronal survival. Recent studies have suggested that the use of astrocytes in an in vitro neurotoxicity test system may prove more relevant to human CNS structure and function than neuronal cells alone. Accordingly, we have developed an HCA assay for co-culture of neurons and astrocytes, comprised of protocols and validated, target-specific detection reagents for profiling &beta;III-tubulin and glial fibrillary acidic protein (GFAP). This assay enables simultaneous analysis of neurotoxicity, neurite outgrowth, gliosis, neuronal and astrocytic morphology and neuronal and astrocytic development in a wide variety of cellular models, representing a novel, non-subjective, high-throughput assay for neurotoxicity assessment. The assay holds great potential for enhanced detection of neurotoxicity and improved productivity in neuroscience research and drug discovery.

This article describes a novel protocol and reagent set designed for sensitive measurement of neurotoxic effects of compounds and treatments on co-cultures of neurons and astrocytes using high content analysis. Results demonstrate that high content analysis represents an exciting novel technology for neurotoxicity assessment.

Nörotoksisite Yüksek İçerik Analizi Nöronal ve Astrosit Hücre Co-Kültür Testi

High Content Analysis (HCA) assays combine cells and detection reagents with automated imaging and powerful image analysis algorithms, allowing ...

Biyoloji

Semi-automated Optical Heartbeat Analysis of Small Hearts

We have developed a method for analyzing high speed optical recordings from Drosophila, zebrafish and embryonic mouse hearts (Fink, et. al., 2009). Our Semi-automatic Optical Heartbeat Analysis (SOHA) uses a novel movement detection algorithm that is able to detect cardiac movements associated with individual contractile and relaxation events. The program provides a host of physiologically relevant readouts including systolic and diastolic intervals, heart rate, as well as qualitative and quantitative measures of heartbeat arrhythmicity. The program also calculates heart diameter measurements during both diastole and systole from which fractional shortening and fractional area changes are calculated. Output is provided as a digital file compatible with most spreadsheet programs. Measurements are made for every heartbeat in a record increasing the statistical power of the output. We demonstrate each of the steps where user input is required and show the application of our methodology to the analysis of heart function in all three genetically tractable heart models.

We have developed a Semi-automated Optical Heartbeat Analysis method (SOHA) for analyzing high speed optical recordings from Drosophila, zebrafish and embryonic mouse hearts. We demonstrate the application of our methodology to the analysis of heart function in fruit fly and embryonic mouse hearts.

Küçük Kalpler Yarı-otomatik Optik Heartbeat Analizi

We have developed a method for analyzing high speed optical recordings from Drosophila, zebrafish and embryonic mouse hearts (Fink, et. al., 2009). Our ...

Transplantation of GFP-expressing Blastomeres for Live Imaging of Retinal and Brain Development in Chimeric Zebrafish Embryos

Cells change extensively in their locations and property during embryogenesis. These changes are regulated by the interactions between the cells and their environment. Chimeric embryos, which are composed of cells of different genetic background, are great tools to study the cell-cell interactions mediated by genes of interest. The embryonic transparency of zebrafish at early developmental stages permits direct visualization of the morphogenesis of tissues and organs at the cellular level. Here, we demonstrate a protocol to generate chimeric retinas and brains in zebrafish embryos and to perform live imaging of the donor cells. The protocol covers the preparation of transplantation needles, the transplantation of GFP-expressing donor blastomeres to GFP-negative hosts, and the examination of donor cell behavior under live confocal microscopy. With slight modifications, this protocol can also be used to study the embryonic development of other tissues and organs in zebrafish. The advantages of using GFP to label donor cells are also discussed. 

We demonstrate a protocol to generate chimeric zebrafish embryos for live imaging cellular behavior during embryogenesis.

Transplantasyon GFP of-Kimerik Zebra balığı Embriyolar Retina ve Beyin Gelişimi Canlı Görüntüleme blastomer ifade

Cells change extensively in their locations and property during embryogenesis. These changes are regulated by the interactions between the cells and their ...

Imaging Glycans in Zebrafish Embryos by Metabolic Labeling and Bioorthogonal Click Chemistry

Imaging glycans in vivo has recently been enabled using a bioorthogonal chemical reporter strategy by treating cells or organisms with azide- or alkyne-tagged monosaccharides1, 2. The modified monosaccharides, processed by the glycan biosynthetic machinery, are incorporated into cell surface glycoconjugates. The bioorthogonal azide or alkyne tags then allow covalent conjugation with fluorescent probes for visualization, or with affinity probes for enrichment and glycoproteomic analysis. This protocol describes the procedures typically used for noninvasive imaging of fucosylated glycans in zebrafish embryos, including: 1) microinjection of one-cell stage embryos with GDP-5-alkynylfucose (GDP-FucAl), 2) labeling fucosylated glycans in the enveloping layer of zebrafish embryos with azide-conjugated fluorophores via biocompatible Cu(I)-catalyzed azide-alkyne cycloaddition (CuAAC), and 3) imaging by confocal microscopy3. The method described here can be readily extended to visualize other classes of glycans, e.g. glycans containing sialic acid4 and N-acetylgalactosamine5, 6, in developing zebrafish and in other living organisms.

A click-chemistry based method that allows for the rapid, noninvasive, and robust labeling of alkyne-tagged glycans in zebrafish embryos is described. Fucosylated glycans in the enveloping layer of zebrafish embryos in the late gastrulation stage were imaged in this study.

Metabolik Etiketleme ve Bioorthogonal tıklayın Kimya Zebra balığı Embriyolar Görüntüleme glukanlardır

Imaging glycans in vivo has recently been enabled using a bioorthogonal chemical reporter strategy by treating cells or organisms with azide- or ...

Quantitative and Automated High-throughput Genome-wide RNAi Screens in C. elegans

RNA interference is a powerful method to understand gene function, especially when conducted at a whole-genome scale and in a quantitative context. In C. elegans, gene function can be knocked down simply and efficiently by feeding worms with bacteria expressing a dsRNA corresponding to a specific gene 1. While the creation of libraries of RNAi clones covering most of the C. elegans genome 2,3 opened the way for true functional genomic studies (see for example 4-7), most established methods are laborious. Moy and colleagues have developed semi-automated protocols that facilitate genome-wide screens 8. The approach relies on microscopic imaging and image analysis. 
	Here we describe an alternative protocol for a high-throughput genome-wide screen, based on robotic handling of bacterial RNAi clones, quantitative analysis using the COPAS Biosort (Union Biometrica (UBI)), and an integrated software: the MBioLIMS (Laboratory Information Management System from Modul-Bio) a technology that provides increased throughput for data management and sample tracking. The method allows screens to be conducted on solid medium plates. This is particularly important for some studies, such as those addressing host-pathogen interactions in C. elegans, since certain microbes do not efficiently infect worms in liquid culture.
	We show how the method can be used to quantify the importance of genes in anti-fungal innate immunity in C. elegans. In this case, the approach relies on the use of a transgenic strain carrying an epidermal infection-inducible fluorescent reporter gene, with GFP under the control of the promoter of the antimicrobial peptide gene nlp 29 and a red fluorescent reporter that is expressed constitutively in the epidermis. The latter provides an internal control for the functional integrity of the epidermis and nonspecific transgene silencing9. When control worms are infected by the fungus they fluoresce green. Knocking down by RNAi a gene required for nlp 29 expression results in diminished fluorescence after infection. Currently, this protocol allows more than 3,000 RNAi clones to be tested and analyzed per week, opening the possibility of screening the entire genome in less than 2 months.

Quantitative and Automated High-throughput Genome-wide RNAi Screens in C. elegans

We describe a protocol using C. elegans and RNAi feeding libraries that allows automated measurement of multiple parameters such as fluorescence, size and opacity of individual worms in a population. We give one example of a screen to identify genes involved in anti-fungal innate immunity in C. elegans.

Nicel ve Otomatik Yüksek throughput Genom çapında RNAi Ekranlar C. elegans</em

RNA interference is a powerful method to understand gene function, especially when conducted at a whole-genome scale and in a quantitative context. In C. ...

Cell Tracking Using Photoconvertible Proteins During Zebrafish Development

Embryogenesis is a dynamic process that is best studied by using techniques that allow the documentation of developmental changes in vivo. The use of genetically-encoded fluorescent proteins has proven a valuable strategy for elucidating dynamic morphogenetic processes as they occur in the intact organism. During the past decade, the development of photoactivatable and photoconvertible fluorescent proteins has opened the possibility to investigate the fate of discrete subpopulations of tagged proteins1. Unlike photoactivatable proteins, photoconvertible fluorescent proteins (PCFPs) are readily tracked and imaged in their native emission state prior to photoconversion, making it easier to identify and select regions by optical inspection. PCFPs, such as Kaede2, KikGR3, Dendra4 and EosFP5, can be shifted from green to red upon exposure to UV or blue light due to a His-Tyr-Gly tripeptide sequence which forms a green chromophore that can be photoconverted to a red one by a light-catalyzed &beta;-elimination and subsequent extension of a &pi;-conjugated system3. PCFPs and their monomeric variants are useful tools for tracking cells6-10 and studying protein dynamics11-14, respectively. During recent years, PCFPs have been expressed in different animal model, such as zebrafish6, chicken7,8 and mouse9,10 for cell fate tracking. Here we report a protocol for cell-specific photoconversion of PCFPs in the living zebrafish embryo and further tracking of photoconverted proteins at later developmental stages. This methodology allows studying, in a tissue-specific manner, cell biological events underlying morphogenesis in the zebrafish animal model.

Here, we present a method for the photoactivated switch of photoconvertible fluorescent proteins (PCFPs) in the living zebrafish embryo and further tracking of photoconverted protein at specific time points during development. This methodology allows monitoring of cell biological events underlying different developmental processes in a live vertebrate organism.

Zebra balığı Geliştirme Sırasında Photoconvertible Proteinler kullanma Cep Takip

Embryogenesis is a dynamic process that is best studied by using techniques that allow the documentation of developmental changes in vivo. The use of ...

Semi-automated Imaging of Tissue-specific Fluorescence in Zebrafish Embryos

Zebrafish embryos are a powerful tool for large-scale screening of small molecules. Transgenic zebrafish that express fluorescent reporter proteins are frequently used to identify chemicals that modulate gene expression. Chemical screens that assay fluorescence in live zebrafish often rely on expensive, specialized equipment for high content screening. We describe a procedure using a standard epifluorescence microscope with a motorized stage to automatically image zebrafish embryos and detect tissue-specific fluorescence. Using transgenic zebrafish that report estrogen receptor activity via expression of GFP, we developed a semi-automated procedure to screen for estrogen receptor ligands that activate the reporter in a tissue-specific manner. In this video we describe procedures for arraying zebrafish embryos at 24-48 hours post fertilization (hpf) in a 96-well plate and adding small molecules that bind estrogen receptors. At 72-96 hpf, images of each well from the entire plate are automatically collected and manually inspected for tissue-specific fluorescence. This protocol demonstrates the ability to detect estrogens that activate receptors in heart valves but not in liver.

Described here is a protocol for semi-automated imaging of tissue-specific fluorescence in zebrafish embryos.

Zebra balığı Embriyolar Doku spesifik Floresans yarı otomatik Görüntüleme

Zebrafish embryos are a powerful tool for large-scale screening of small molecules. Transgenic zebrafish that express fluorescent reporter proteins are ...

Open Source High Content Analysis Utilizing Automated Fluorescence Lifetime Imaging Microscopy

We present an open source high content analysis instrument utilizing automated fluorescence lifetime imaging (FLIM) for assaying protein interactions using F&#246;rster resonance energy transfer (FRET) based readouts of fixed or live cells in multiwell plates. This provides a means to screen for cell signaling processes read out using intramolecular FRET biosensors or intermolecular FRET of protein interactions such as oligomerization or heterodimerization, which can be used to identify binding partners. We describe here the functionality of this automated multiwell plate FLIM instrumentation and present exemplar data from our studies of HIV Gag protein oligomerization and a time course of a FRET biosensor in live cells. A detailed description of the practical implementation is then provided with reference to a list of hardware components and a description of the open source data acquisition software written in &#181;Manager. The application of FLIMfit, an open source MATLAB-based client for the OMERO platform, to analyze arrays of multiwell plate FLIM data is also presented. The protocols for imaging fixed and live cells are outlined and a demonstration of an automated multiwell plate FLIM experiment using cells expressing fluorescent protein-based FRET constructs is presented. This is complemented by a walk-through of the data analysis for this specific FLIM FRET data set.

We present an open source high content analysis (HCA) instrument utilizing automated fluorescence lifetime imaging (FLIM) for assaying protein interactions using F&#246;rster resonance energy transfer (FRET) based readouts. Data acquisition for this openFLIM-HCA instrument is controlled by software written in &#181;Manager and data analysis is undertaken in FLIMfit.

Otomatik Flüoresans Ömrü Görüntüleme Mikroskopi kullanmak Open Source Yüksek İçerik Analizi

We present an open source high content analysis instrument utilizing automated fluorescence lifetime imaging (FLIM) for assaying protein interactions ...

Analysis of Beta-cell Function Using Single-cell Resolution Calcium Imaging in Zebrafish Islets

Pancreatic beta-cells respond to increasing blood glucose concentrations by secreting the hormone insulin. The dysfunction of beta-cells leads to hyperglycemia and severe, life-threatening consequences. Understanding how the beta-cells operate under physiological conditions and what genetic and environmental factors might cause their dysfunction could lead to better treatment options for diabetic patients. The ability to measure calcium levels in beta-cells serves as an important indicator of beta-cell function, as the influx of calcium ions triggers insulin release. Here we describe a protocol for monitoring the glucose-stimulated calcium influx in zebrafish beta-cells by using GCaMP6s, a genetically encoded sensor of calcium. The method allows monitoring the intracellular calcium dynamics with single-cell resolution in ex vivo mounted islets. The glucose-responsiveness of beta-cells within the same islet can be captured simultaneously under different glucose concentrations, which suggests the presence of functional heterogeneity among zebrafish beta-cells. Furthermore, the technique provides high temporal and spatial resolution, which reveals the oscillatory nature of the calcium influx upon glucose stimulation. Our approach opens the doors to use the zebrafish as a model to investigate the contribution of genetic and environmental factors to beta-cell function and dysfunction.

Beta-cell functionality is important for blood-glucose homeostasis, which is evaluated at single-cell resolution using a genetically encoded reporter for calcium influx.

Zebra balığı adacıkları tek hücreli çözünürlük kalsiyum Imaging'i kullanma Beta hücre fonksiyonu Analizi

Pancreatic beta-cells respond to increasing blood glucose concentrations by secreting the hormone insulin. The dysfunction of beta-cells leads to ...

Automated Detection and Analysis of Exocytosis

Timelapse TIRF microscopy of pH-sensitive GFP (pHluorin) attached to vesicle SNARE proteins is an effective method to visualize single vesicle exocytic events in cell culture. To perform an unbiased, efficient identification and analysis of such events, a computer-vision based approach was developed and implemented in MATLAB. The analysis pipeline consists of a cell segmentation and exocytic-event identification algorithm. The computer-vision approach includes tools for investigating multiple parameters of single events, including the half-life of fluorescence decay and peak &#916;F/F, as well as whole-cell analysis of the frequency of exocytosis. These and other parameters of fusion are used in a classification approach to distinguish distinct fusion modes. Here a newly built GUI performs the analysis pipeline from start to finish. Further adaptation of Ripley&#39;s K function in R Studio is used to distinguish between clustered, dispersed, or random occurrence of fusion events in both space and time.

We developed automated computer vision software to detect exocytic events marked by pH-sensitive fluorescent probes. Here, we demonstrate the use of a graphical user interface and RStudio to detect fusion events, analyze and display spatiotemporal parameters of fusion, and classify events into distinct fusion modes.

Eksositozun Otomatik Tespiti ve Analizi

Timelapse TIRF microscopy of pH-sensitive GFP (pHluorin) attached to vesicle SNARE proteins is an effective method to visualize single vesicle exocytic ...