Biz minnetle ve laboratuvar desteği ve yararlı tartışmalar için Yaşlılık ve Beslenme (CISBAN) Entegre Sistem Biyolojisi Merkezi&#39;nin tüm üyeleri kabul ediyorsunuz. Bu çalışma, Biyoteknoloji ve Biyolojik Bilimler Araştırma Konseyi (BBSRC) (BB/C008200/1) ve Wellcome Trust (075.294, 093.088) tarafından desteklenmiştir.

<ol>
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Biotechnol. 22 (1), 94-102 (2011).</li><li>Maringele, L., Lydall, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=EXO1-dependent+single-stranded+DNA+at+telomeres+activates+subsets+of+DNA+damage+and+spindle+checkpoint+pathways+in+budding+yeast+yku70&#916;mutants.">EXO1-dependent single-stranded DNA at telomeres activates subsets of DNA damage and spindle checkpoint pathways in budding yeast yku70&#916;mutants.</a> Genes and Dev. 16 (15), 1919-1933 (2002).</li><li>Warringer, J., Blomberg, 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+screening+in+environmental+arrays+allows+analysis+of+quantitative+phenotypic+profiles+in+Saccharomyces+cerevisiae.">Automated screening in environmental arrays allows analysis of quantitative phenotypic profiles in Saccharomyces cerevisiae.</a> Yeast. 20, 53-67 (2003).</li><li>Hillenmeyer, M., Fung, E., Wildenhain, J., Pierce, S., Hoon, S., Lee, W., Proctor, M., St. Onge, R., Tyers, M., Koller, D., Altman, R., Davis, R., Nislow, C., Giaever, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+chemical+genomic+portrait+of+yeast:+uncovering+a+phenotype+for+all+genes.">The chemical genomic portrait of yeast: uncovering a phenotype for all genes.</a> Science. 320 (5874), 362-365 (2008).</li><li>Smith, A. M., Durbic, T., Oh, J., Urbanus, M., Proctor, M., Heisler, L. E., Giaever, G., Nislow, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Competitive+Genomic+Screens+of+Barcoded+Yeast+Libraries.">Competitive Genomic Screens of Barcoded Yeast Libraries.</a> J. Vis. Exp. (54), e2864 (2011).</li><li>Lederberg, J., Lederberg, E. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Replica+plating+and+indirect+selection+of+bacterial+mutants.">Replica plating and indirect selection of bacterial mutants.</a> J. 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Çıkar çatışması ilan etti.

QFA birçok bakımdan bir doğrudan üç kurulan tezgah ölçekli mikrobiyolojik tekniklerin soyundan: kültür dilüsyon serisi nokta testleri 9, gazlı sıvı kültürleri 9 ve çoğaltma kaplama 13 büyüme eğrisi belirlenmesi. Bu üç yöntem özetlenmiş ve QFA ve Tablo 2&#39;de diğer yüksek verimlilik teknikleri ile karşılaştırılmıştır. Dilüsyon serisi nokta testleri inokulum dilüsyonları bir dizi yetiştirilen kültürlerin bir dizi koloniler kurma yeteneğini bir zorlanma fitness tanımlamak Oysa, tek bir kültür tekrarlanan gözlemlere QFA önlemler gerginlik spor bir büyüme eğrisi inşa etmek. Tek bir kültürden spor miktarının daha fazla sayıda suşları aynı koşullar altında, eş zamanlı olarak incelenmiştir izin verir. Farklı ortamlarda veya genetik kökenli kültür spor farklılıkların test ederken kültürlerin çoğaltılıyor diziler çoğu yararlı suşu koleksiyonları, tekrarlanan test yapılmasını sağlar. QFA protokolü sunduBurada, bağımsız kültürleri (robotik QFA, Tablo 2) binlerce büyüme gözlemlemek için uygun inkübe ve görüntü agar, çoğaltmak aşılamak için pahalı robotik ekipman kullanır. QFA köklü, geleneksel laboratuvar teknikleri dayanmaktadır Bununla birlikte, aynı zamanda manuel adımları (manuel QFA, Tablo 2) robot yardımı yerine daha ucuza çok daha yapılabilir. Manuel QFA manuel pin aracı ve ve fotoğraflar için inkübatör plakalarını manuel aktarma yöntemi kullanarak agar plaklarına kültürlerin çoğaltma ve aşılama içerir. Aynı bilişimsel analizi akışı ya deney tasarımı gelen büyüme eğrilerinin üretmek için uygulanabilir. QFA spor tahminleri nispeten kesin gibi görünüyor. Şekil 4&#39;te, işlevsel olarak ilgili genin delesyonları dört örneğin kümelerinin ortalama uyumu vurgulanmıştır. İki bağımsızlık her işlevsel ilişkili gen sınıfın üyelerinin yakınent genetik kökenden (ura3Δ ve cdc13-1) QFA spor tahminlerinin tekrarlanabilirlik gösterir. Örneğin, sadece 3 gen delesyonu (dışarı olası 4.300 ile) korunmuş MRX kompleksinin üç üyesi ayırın. Mikrobik suşların büyüme özellikleri test etmek için QFA Yüksek throughput alternatifler SGA 5,6, spektrofotometrik plaka okuyucuları optik yoğunluk kinetik yakalayarak rekabet barkodlu kütüphanelerde ekranlar 11,12 ve ekranlar dahil Tablo 2&#39;de ve daha tam olarak başka bir yerde 8 açıklanan 10 . QFA ve kültürler katı agar yüzeyler (agar temelli yöntemler, Tablo 2) büyümek SGA tabak, robot tarafından hızlı ve kolay işlenebilir. Katı agar yüzeyinden kültürleri de büyüme boyunca havalandırılmakta ve hücrelerin sosyal mikroplar bir topluluk 14 büyürse, sabit kültürlerde büyüyebilir. Sol bozulmamış, mikrobiyal topluluklar birkendi mikro-ortam, örneğin etanol gibi salgılayan toksinler, ve muhtemelen hücreleri arasındaki sinyal ffect. Ancak, yeterli havalandırma sağlamak için gerekli sıvı kültürleri, karıştırma sürekli, yapay büyüme tarzlarını etkileyebilir mikrobiyal topluluklar ve onların mikro-ortamlar bozar. Kültürler arasındaki hücreleri taşıyan sıvı sıçraması için daha az fırsat olmadığı için çapraz kontaminasyon katı agar yöntemleri daha az kaygı verici olduğunu. Kontaminasyon katı agar tayinlerde yabancı hava yoluyla bulaşan mikroplar tarafından meydana gelirse, genellikle katı agar görsel muayene ile tespit ve hesaba veya kaldırılabilir. QFA üretilen iki şekilde paralel sıvı yöntemlerine göre daha yüksektir. Öncelikle, QFA (ve SGA) tabaklarda, inoküle kültürleri plaka başına daha bağımsız kültür vererek, daha yoğun birlikte paketlenmiştir. QFA yılında 308 kültürleri deneysel olmayan kenar kültürler (Şekil 1) com saymazsak, genellikle vardırplaka başına 96 veya 100 kültürleri ile sıvı kültür paralel olarak hazırlandık. İkincisi, QFA deneyler plakaların çok daha büyük bir sayıya ölçeklendirilebilir. Tek bir QFA deneyde analiz plaka sayıda tek bir inkübatör ya sıcak bir odada, en az kabul edilebilir görüntü yakalama frekansı ve maksimum elde yakalama frekansı mevcut alan ile sınırlanmıştır ise, bir sıvı büyüme deneyde plakaların numarası kuvvetli sınırlıdır plaka okuyucu (tipik olarak bir ya da iki levha) veya plak okuyucu (tipik olarak 25-50 plakaları) eklenmiş istif bölmesi arasında kapasitesiyle. Yakın zamanda 37.884 eşzamanlı kültürleri vererek, 308 deneysel kültürleri olan 123 tabak, her bir tam otomatik QFA deney gerçekleştirdi. Biz sıvı büyüyen bağımsız kültürlerin maksimum elde edilebilir sayısı 96 (kültür / plaka) olduğu tahmin bizim QFA verimi az on misli civarındadır x 50 (tabak / istifleyici) = 4800. Sıvı kültür ekranları için alternatif bir çok otomatikleştirilmiş gro kullanmaktırparalel wth cihazları (8 Blomberg tarafından incelemesi ile Tablo 1), bir ya da iki levha bir kapasiteye sahip her. Her cihaz Sıcaklık kontrolü bağımsızdır ve bu nedenle şartlar aynı değildir, ama onlar olduğunu varsayarak, bu iş en az 190 tür cihazların QFA throughput (cihaz başına 200 sıvı kültürleri varsayarak) maç gerektirecektir. Özetle, QFA yararlı küçük ölçekli odaklanmış deneyler ve yüksek işlem ekranlar hem de nicel büyüme fenotipleri toplamak için uygulanabilir yüksek kaliteli bir iş akışıdır. Robotik ekipman için gereksinimleri değişen etkili uygulanacak esnektir. QFA iş akışının bilgisayar bileşeni serbestçe kullanılabilir, açık kaynak kodu dayanmaktadır.

<table border="1"><tbody><tr><td> Reaktif / ekipman Adı </td><td> Şirket </td><td> Katalog numarası </td><td> Yorumlar </td></tr><tr><td> Replica Plater </td><td> Sigma </td><td> R-2508 </td><td> 1/8 &quot;çaplı iğneler ile 96 pin manuel pintool </td></tr><tr><td> Mate Mix </td><td> Eppendorf </td><td> 5353 000.014 </td><td></td></tr><tr><td> Biomek FX </td><td> Beckman </td><td> A31842 </td><td></td></tr><tr><td> Teleshake </td><td> Thermo Scientific </td><td> 50095890 </td><td> Biomek FX Yüklendi </td></tr><tr><td> BM3-SC </td><td> S &amp; P Robotik A.Ş. </td><td> BM3-SC </td><td> 192 raf dönen atlıkarınca </td></tr><tr><td> spImager </td><td> S &amp; P Robotik A.Ş. </td><td> spImager </td><td> Yüksek çözünürlüklü görüntüleme kılavuzu </td></tr><tr><td> sitometride ile spImagerat </td><td> S &amp; P Robotik A.Ş. </td><td> Özel </td><td> Sıcaklık otomatik yüksek çözünürlüklü görüntüleme kontrol </td></tr><tr><td> Isı değiştirici ile Cytomat 6001 </td><td> Thermo Scientific </td><td> 51022222 </td><td> SpImager Bağlı </td></tr><tr><td> Ecoline RE207 </td><td> Lauda </td><td> RE207 </td><td> Cytomat Bağlı </td></tr><tr><td> atlıkarınca ile spImager </td><td> S &amp; P Robotik A.Ş. </td><td> Özel </td><td> Otomatik yüksek çözünürlüklü görüntüleme </td></tr><tr><td> FP12pins için Robot pin aracı fikstürü </td><td> V &amp; P Bilimsel </td><td> AFIX96FP12 </td><td> N / A </td></tr><tr><td> 96 x FP12 pimleri </td><td> V &amp; P Bilimsel </td><td> FP12 </td><td> 50.4 mm uzunluğunda, 17 mm pin uzunluğunun maruz </td></tr><tr><td> Pin Aracı İstasyonu Yerleştirme </td><td> V &amp; P Bilimsel </td><td> VP425 </td><td> Docking istasyonu baz pin fan kurutma izin kaldırıldı </td></tr><tr><td>Temizleme Fırçası Pin </td><td> V &amp; P Bilimsel </td><td> VP425 </td><td> N / A </td></tr><tr><td> Replica Plater </td><td> Sigma </td><td> R-2508 </td><td> Manuel pin aracı </td></tr><tr><td> Kapaklı Nunc OmniTray </td><td> Nunc </td><td> 734-0490 </td><td> N / A </td></tr><tr><td> 96 steril polistiren levhalar </td><td> Greiner BioOne </td><td> 655161 </td><td> N / A </td></tr><tr><td> 96 steril polistiren kapakları </td><td> Greiner BioOne </td><td> 656171 </td><td> N / A </td></tr></tbody></table> Tablo 3. Özel reaktifleri ve ekipmanları.

a quantitative fitness analysis workflow

Kantitatif Spor Analizi (QFA) paralel 1,2,3,4 yetiştirilen mikrobiyal kültürlerin uyumu karşılaştırmak için bir deney ve hesaplama iş akışıdır. QFA tek kültür odaklı gözlem uygulandı, ancak bağımsız kültürlerin binlerce kadar araştıran genom genetik etkileşim veya ilaç ekranlar için en yararlı olabilir. Merkezi deneysel yöntem inkübe ve düzenli çekilmiştir katı agar plaklarına bağımsız, seyreltik sıvı mikrobiyal kültürlerin aşılama olduğunu. Her zaman noktasından fotoğraflar nicel spor önlemler elde izin vererek, büyüme eğrileri oluşturmak için kullanılır kantitatif hücre yoğunluğu tahminleri, üretim, analiz edilir. Kültür uyumu ölçmek ve genetik etkileşimin güçlü veya ilaç hassasiyetleri rütbe karşılaştırılabilir. Herhangi bir tedavi kültür fitness üzerinde etkisi substrat agar (örneğin küçük moleküller, antibiyotikler veya besin) olarak eklenir veya plakaları dışarıdan uygulananly (örn. UV radyasyon, sıcaklık) QFA ile belirlenebilir. QFA iş akışı büyüme oranı 96-veya 200-iyi plaka okuyucuları paralel sıvı kültürleri spektrofotometrik ölçülmesi ile elde edilen benzer tahmin üretir. Önemlisi, QFA tür yöntemleri ile karşılaştırıldığında daha yüksek hızı daha vardır. QFA kültürleri sağlam bir agar yüzeyinde büyür ve bu nedenle de Karıştıktan gerek kalmadan büyüme sırasında havalandırılmakta. QFA üretilen bazı Sentetik Genetik Array (SGA) tarama yöntemleri 5,6 o kadar yüksek değildir. QFA kültürlerin yoğun agara inoküle edilmeden önce seyreltilir beri Ancak, QFA 3 üstel ve doygunluk aşamaları da dahil olmak üzere daha kapsamlı büyüme eğrileri, yakalayabilir. Örneğin, büyüme eğrisi gözlemleri daha önce 1 anlatıldığı gibi kültür katlama süreleri, yüksek hassasiyetli doğrudan tahmin edilmesine olanak tanımaktadır. Burada bir sunuyoruzBelirli QFA protokol S. binlerce uygulanan otomatik inokülasyon, inkübasyon ve görüntüleme sırasında robotlar tarafından işlenir cerevisiae kültürleri. Bu otomatik adımları herhangi üretilen bir azalma ile ilişkilendirilmiş, bir eşdeğer, manuel prosedürü ile değiştirilir, ve bu da daha düşük bir verim kılavuzu protokolü mevcut olabilir. Aynı QFA yazılım araçları ya da iş akışı içinde çekilen resimlere uygulanabilir. Biz geniş bir deneyime tomurcuklanan maya kültürleri QFA uygulayarak var S. ama biz QFA fisyon maya kültürleri incelemek için eşit derecede yararlı olacağını bekliyoruz S. cerevisiae pombe ve bakteri kültürleri.

Watch this Scientific Journal Video about A Quantitative Fitness Analysis Workflow at JoVE.com

A Quantitative Fitness Analysis Workflow

Kantitatif Spor Analizi (QFA) mikrobiyal kültür uyumu tahmin etmek için deneysel ve hesaplamalı yöntemleri tamamlayıcı bir dizi. QFA genetik mutasyonlar, uyuşturucu ya da mikrop büyümesi üzerine uygulanan diğer tedavilerin etkisi tahmin ediyor. Tek kültür odaklı analiz paralel kültürlerin binlerce deneyler ölçekleme dizayn edilebilir.

Kantitatif Spor Analizi İş Akışı

Quantitative Fitness Analysis (QFA) is an experimental and computational workflow for comparing fitnesses of microbial cultures grown in parallel1,2,3,4. ...

Research

JoVE Journal

Biology

14.3K Görüntüleme.  Newcastle University Medical School. Kantitatif Spor Analizi (QFA) mikrobiyal kültür uyumu tahmin etmek için deneysel ve hesaplamalı yöntemleri tamamlayıcı bir dizi. QFA genetik mutasyonlar, uyuşturucu ya da mikrop büyümesi üzerine uygulanan diğer tedavilerin etkisi tahmin ediyor. Tek kültür odaklı analiz paralel kültürlerin binlerce deneyler ölçekleme dizayn edilebilir.

Video: A Quantitative Fitness Analysis Workflow

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

Quantitative Phosphoproteomics in Fatty Acid Stimulated Saccharomyces cerevisiae

This protocol describes the growth and stimulation, with the fatty acid oleate, of isotopically heavy and light S. cerevisiae cells. Cells are ground using a cryolysis procedure in a ball mill grinder and the resulting grindate brought into solution by urea solubilization. This procedure allows for the lysis of the cells in a metabolically inactive state, preserving phosphorylation and preventing reorientation of the phosphoproteome during cell lysis.  Following reduction, alkylation, trypsin digestion of the proteins, the samples are desalted on C18 columns and the sample complexity reduced by fractionation using hydrophilic interaction chromatography (HILIC). HILIC columns preferentially retain hydrophilic molecules which is well suited for phosphoproteomics.  Phosphorylated peptides tend to elute later in the chromatographic profile than the non phosphorylated counterparts. After fractionation, phosphopeptides are enriched using immobilized metal chromatography, which relies on charge-based affinities for phosphopeptide enrichment. At the end of this procedure the samples are ready to be quantitatively analyzed by mass spectrometry.

Quantitative Phosphoproteomics in Fatty Acid Stimulated Saccharomyces cerevisiae

Description of a quantitative phosphorylation procedure using cryolysis, urea solubilziation, HILIC fractionation and IMAC enrichment of phosphorylated peptides.

Yağ Asidi Kantitatif Phosphoproteomics Uyarılmış Saccharomyces cerevisiae</em

This protocol describes the growth and stimulation, with the fatty acid oleate, of isotopically heavy and light S. cerevisiae cells. Cells are ground ...

Live-cell Imaging and Quantitative Analysis of Embryonic Epithelial Cells in Xenopus laevis

Embryonic epithelial cells serve as an ideal model to study morphogenesis where multi-cellular tissues undergo changes in their geometry, such as changes in cell surface area and cell height, and where cells undergo mitosis and migrate. Furthermore, epithelial cells can also regulate morphogenetic movements in adjacent tissues1. A traditional method to study epithelial cells and tissues involve chemical fixation and histological methods to determine cell morphology or localization of particular proteins of interest. These approaches continue to be useful and provide "snapshots" of cell shapes and tissue architecture, however, much remains to be understood about how cells acquire specific shapes, how various proteins move or localize to specific positions, and what paths cells follow toward their final differentiated fate. High resolution live imaging complements traditional methods and also allows more direct investigation into the dynamic cellular processes involved in the formation, maintenance, and morphogenesis of multicellular epithelial sheets. Here we demonstrate experimental methods from the isolation of animal cap tissues from Xenopus laevis embryos to confocal imaging of epithelial cells and simple measurement approaches that together can augment molecular and cellular studies of epithelial morphogenesis.

Live-cell Imaging and Quantitative Analysis of Embryonic Epithelial Cells in Xenopus laevis

Xenopus embryonic epithelia are an ideal model system to study cell behaviors such as polarity development and shape change during epithelial morphogenesis. Traditional histology of fixed samples is increasingly being complemented by live-cell confocal imaging. Here we demonstrate methods to isolate frog tissues and visualize live epithelial cells and their cytoskeleton using live-cell confocal microscopy.

Canlı hücre Görüntüleme ve Kantitatif Analiz Embriyonik Epitel Hücreleri Xenopus laevis</em

Embryonic epithelial cells serve as an ideal model to study morphogenesis where multi-cellular tissues undergo changes in their geometry, such as changes ...

Quantitative Live Cell Fluorescence-microscopy Analysis of Fission Yeast

Several microscopy techniques are available today that can detect a specific protein within the cell. During the last decade live cell imaging using fluorochromes like Green Fluorescent Protein (GFP) directly attached to the protein of interest has become increasingly popular 1. Using GFP and similar fluorochromes the subcellular localisations and movements of proteins can be detected in a fluorescent microscope. Moreover, also the subnuclear localisation of a certain region of a chromosome can be studied using this technique. GFP is fused to the Lac Repressor protein (LacR) and ectopically expressed in the cell where tandem repeats of the lacO sequence has been inserted into the region of interest on the chromosome2. The LacR-GFP will bind to the lacO repeats and that area of the genome will be visible as a green dot in the fluorescence microscope. Yeast is especially suited for this type of manipulation since homologous recombination is very efficient and thereby enables targeted integration of the lacO repeats and engineered fusion proteins with GFP 3. Here we describe a quantitative method for live cell analysis of fission yeast. Additional protocols for live cell analysis of fission yeast can be found, for example on how to make a movie of the meiotic chromosomal behaviour 4. In this particular experiment we focus on subnuclear organisation and how it is affected during gene induction. We have labelled a gene cluster, named Chr1, by the introduction of lacO binding sites in the vicinity of the genes. The gene cluster is enriched for genes that are induced early during nitrogen starvation of fission yeast 5. In the strain the nuclear membrane (NM) is labelled by the attachment of mCherry to the NM protein Cut11 giving rise to a red fluorescent signal. The Spindle Pole body (SPB) compound Sid4 is fused to Red Fluorescent Protein (Sid4-mRFP) 6. In vegetatively growing yeast cells the centromeres are always attached to the SPB that is embedded in the NM 7. The SPB is identified as a large round structure in the NM. By imaging before and 20 minutes after depletion of the nitrogen source we can determine the distance between the gene cluster (GFP) and the NM/SPB. The mean or median distances before and after nitrogen depletion are compared and we can thus quantify whether or not there is a shift in subcellular localisation of the gene cluster after nitrogen depletion.

The fission yeast, Schizosaccharomyces pombe, is a good model system to study basic cellular processes. Here we describe a method to perform quantitative live cell analysis of fission yeast. In this particular experiment we focus on organisation of the genome within the cell nucleus, but the method can also be used to study cytosolic factors.

Fisyon Maya Kantitatif Canlı Hücre Floresan mikroskopi Analizi

Several microscopy techniques are available today that can detect a specific protein within the cell. During the last decade live cell imaging using ...

Quantitative Analysis of Random Migration of Cells Using Time-lapse Video Microscopy

Cell migration is a dynamic process, which is important for embryonic development, tissue repair, immune system function, and tumor invasion 1, 2. During directional migration, cells move rapidly in response to an extracellular chemotactic signal, or in response to intrinsic cues 3 provided by the basic motility machinery. Random migration occurs when a cell possesses low intrinsic directionality, allowing the cells to explore their local environment.
Cell migration is a complex process, in the initial response cell undergoes polarization and extends protrusions in the direction of migration 2. Traditional methods to measure migration such as the Boyden chamber migration assay is an easy method to measure chemotaxis in vitro, which allows measuring migration as an end point result. However, this approach neither allows measurement of individual migration parameters, nor does it allow to visualization of morphological changes that cell undergoes during migration.
Here, we present a method that allows us to monitor migrating cells in real time using video - time lapse microscopy. Since cell migration and invasion are hallmarks of cancer, this method will be applicable in studying cancer cell migration and invasion in vitro. Random migration of platelets has been considered as one of the parameters of platelet function 4, hence this method could also be helpful in studying platelet functions. This assay has the advantage of being rapid, reliable, reproducible, and does not require optimization of cell numbers. In order to maintain physiologically suitable conditions for cells, the microscope is equipped with CO2 supply and temperature thermostat. Cell movement is monitored by taking pictures using a camera fitted to the microscope at regular intervals. Cell migration can be calculated by measuring average speed and average displacement, which is calculated by Slidebook software.

This method allows monitoring of cells in real time and quantitative measurements of different cell migration parameters such as speed, displacement, and velocity. Unlike the traditional methods, this real time approach is not based on endpoint quantitative migration measurements; instead it allows monitoring and calculating different parameters continuously.

Time-lapse video Mikroskop ile Hücrelerinin Rastgele Göç Kantitatif Analiz

Cell migration is a dynamic process, which is important for embryonic development, tissue repair, immune system function, and tumor invasion 1, 2. During ...

Quantitative Analysis of Autophagy using Advanced 3D Fluorescence Microscopy

Prostate cancer is the leading form of malignancies among men in the U.S. While surgery carries a significant risk of impotence and incontinence, traditional chemotherapeutic approaches have been largely unsuccessful. Hormone therapy is effective at early stage, but often fails with the eventual development of hormone-refractory tumors. We have been interested in developing therapeutics targeting specific metabolic deficiency of tumor cells. We recently showed that prostate tumor cells specifically lack an enzyme (argininosuccinate synthase, or ASS) involved in the synthesis of the amino acid arginine1. This condition causes the tumor cells to become dependent on exogenous arginine, and they undergo metabolic stress when free arginine is depleted by arginine deiminase (ADI)1,10. Indeed, we have shown that human prostate cancer cells CWR22Rv1 are effectively killed by ADI with caspase-independent apoptosis and aggressive autophagy (or macroautophagy)1,2,3. Autophagy is an evolutionarily-conserved process that allows cells to metabolize unwanted proteins by lysosomal breakdown during nutritional starvation4,5. Although the essential components of this pathway are well-characterized6,7,8,9, many aspects of the molecular mechanism are still unclear - in particular, what is the role of autophagy in the death-response of prostate cancer cells after ADI treatment? In order to address this question, we required an experimental method to measure the level and extent of autophagic response in cells - and since there are no known molecular markers that can accurately track this process, we chose to develop an imaging-based approach, using quantitative 3D fluorescence microscopy11,12.
Using CWR22Rv1 cells specifically-labeled with fluorescent probes for autophagosomes and lysosomes, we show that 3D image stacks acquired with either widefield deconvolution microscopy (and later, with super-resolution, structured-illumination microscopy) can clearly capture the early stages of autophagy induction. With commercially available digital image analysis applications, we can readily obtain statistical information about autophagosome and lysosome number, size, distribution, and degree of colocalization from any imaged cell. This information allows us to precisely track the progress of autophagy in living cells and enables our continued investigation into the role of autophagy in cancer chemotherapy.

Autophagy is a ubiquitous process that enables cells to degrade and recycle proteins and organelles. We apply advanced fluorescence microscopy to visualize and quantify the small, but essential, physical changes associated with the induction of autophagy, including the formation and distribution of autophagosomes and lysosomes, and their fusion into autolysosomes.

Gelişmiş 3D Floresan Mikroskobu kullanılarak Otofaji Kantitatif Analiz

Prostate cancer is the leading form of malignancies among men in the U.S. While surgery carries a significant risk of impotence and incontinence, ...

Visualization and Analysis of mRNA Molecules Using Fluorescence In Situ Hybridization in Saccharomyces cerevisiae

The Fluorescence in situ Hybridization (FISH) method allows one to detect nucleic acids in the native cellular environment. Here we provide a protocol for using FISH to quantify the number of mRNAs in single yeast cells. Cells can be grown in any condition of interest and then fixed and made permeable. Subsequently, multiple single-stranded deoxyoligonucleotides conjugated to fluorescent dyes are used to label and visualize mRNAs. Diffraction-limited fluorescence from single mRNA molecules is quantified using a spot-detection algorithm to identify and count the number of mRNAs per cell. While the more standard quantification methods of northern blots, RT-PCR and gene expression microarrays provide information on average mRNAs in the bulk population, FISH facilitates both the counting and localization of these mRNAs in single cells at single-molecule resolution.

Visualization and Analysis of mRNA Molecules Using Fluorescence In Situ Hybridization in Saccharomyces cerevisiae

This protocol describes an experimental procedure for performing Fluorescence in situ Hybridization (FISH) for counting mRNAs in single cells at single-molecule resolution.

Floresan kullanarak mRNA Moleküllerin Görselleştirme ve Analiz<em&gt; Yerinde</emIn&gt; Hibridizasyon<em&gt; Saccharomyces cerevisiae</em

The Fluorescence in situ Hybridization (FISH) method allows one to detect nucleic acids in the native cellular environment. Here we provide a protocol for ...

A High Throughput MHC II Binding Assay for Quantitative Analysis of Peptide Epitopes

Biochemical assays with recombinant human MHC II molecules can provide rapid, quantitative insights into immunogenic epitope identification, deletion, or design1,2. Here, a peptide-MHC II binding assay is scaled to 384-well format. The scaled down protocol reduces reagent costs by 75% and is higher throughput than previously described 96-well protocols1,3-5. Specifically, the experimental design permits robust and reproducible analysis of up to 15 peptides against one MHC II allele per 384-well ELISA plate. Using a single liquid handling robot, this method allows one researcher to analyze approximately ninety test peptides in triplicate over a range of eight concentrations and four MHC II allele types in less than 48 hr. Others working in the fields of protein deimmunization or vaccine design and development may find the protocol to be useful in facilitating their own work. In particular, the step-by-step instructions and the visual format of JoVE should allow other users to quickly and easily establish this methodology in their own labs.

Biochemical assays with recombinant human MHC II molecules can provide rapid, quantitative insights into immunogenic epitope identification, deletion, or design. &#160;Here, a peptide-MHC II binding assay scaled to 384-well plates is described. This cost effective format should prove useful in the fields of protein deimmunization and vaccine design and development.

Peptid Epitopların Kantitatif Analiz için bir yüksek veri akışı MHC II Bağlanma Deneyi

Biochemical assays with recombinant human MHC II molecules can provide rapid, quantitative insights into immunogenic epitope identification, deletion, or ...

A New Technique for Quantitative Analysis of Hair Loss in Mice Using Grayscale Analysis

Alopecia is a common form of hair loss which can occur in many different conditions, including male-pattern hair loss, polycystic ovarian syndrome, and alopecia areata. Alopecia can also occur as a side effect of chemotherapy in cancer patients. In this study, our goal was to develop a consistent and reliable method to quantify hair loss in mice, which will allow investigators to accurately assess and compare new therapeutic approaches for these various forms of alopecia. The method utilizes a standard gel imager to obtain and process images of mice, measuring the light absorption, which occurs in rough proportion to the amount of black (or gray) hair on the mouse. Data that has been quantified in this fashion can then be analyzed using standard statistical techniques (i.e., ANOVA, T-test). This methodology was tested in mouse models of chemotherapy-induced alopecia, alopecia areata and alopecia from waxing. In this report, the detailed protocol is presented for performing these measurements, including validation data from C57BL/6 and C3H/HeJ strains of mice. This new technique offers a number of advantages, including relative simplicity of application, reliance on equipment which is readily available in most research laboratories, and applying an objective, quantitative assessment which is more robust than subjective evaluations. Improvements in quantification of hair growth in mice will improve study of alopecia models and facilitate evaluation of promising new therapies in preclinical studies.

Alopecia is a common form of hair loss which can occur in many different conditions, including as a side-effect of chemotherapy. We have developed a method to quantify hair loss in mice, utilizing a standard gel imager to perform a grayscale analysis, to facilitate study of promising new alopecia therapies.

Gri tonlama Analizi İle Farelerde Saç Dökülmesi Kantitatif Analiz Yeni Bir Teknik

Alopecia is a common form of hair loss which can occur in many different conditions, including male-pattern hair loss, polycystic ovarian syndrome, and ...

Complete Workflow for Analysis of Histone Post-translational Modifications Using Bottom-up Mass Spectrometry: From Histone Extraction to Data Analysis

Nucleosomes are the smallest structural unit of chromatin, composed of 147 base pairs of DNA wrapped around an octamer of histone proteins. Histone function is mediated by extensive post-translational modification by a myriad of nuclear proteins. These modifications are critical for nuclear integrity as they regulate chromatin structure and recruit enzymes involved in gene regulation, DNA repair and chromosome condensation. Even though a large part of the scientific community adopts antibody-based techniques to characterize histone PTM abundance, these approaches are low throughput and biased against hypermodified proteins, as the epitope might be obstructed by nearby modifications. This protocol describes the use of nano liquid chromatography (nLC) and mass spectrometry (MS) for accurate quantification of histone modifications. This method is designed to characterize a large variety of histone PTMs and the relative abundance of several histone variants within single analyses. In this protocol, histones are derivatized with propionic anhydride followed by digestion with trypsin to generate peptides of 5 - 20 aa in length. After digestion, the newly exposed N-termini of the histone peptides are derivatized to improve chromatographic retention during nLC-MS. This method allows for the relative quantification of histone PTMs spanning four orders of magnitude.

This protocol outlines a fully integrated workflow for characterizing histone post-translational modifications using mass spectrometry (MS). The workflow includes histone purification from cell cultures or tissues, histone derivatization and digestion, MS analysis using nano-flow liquid chromatography and instructions for data analysis. The protocol is designed for completion within 2 - 3 days.

Histon Ekstraksiyon Gönderen Data Analizi: Aşağıdan yukarıya Kütle Spektrometre kullanarak histon post-translasyonel modifikasyonlar Analizi için komple iş akışı

Nucleosomes are the smallest structural unit of chromatin, composed of 147 base pairs of DNA wrapped around an octamer of histone proteins. Histone ...