Name: single-cell analysis of bacillus subtilis biofilms using fluorescence microscopy and flow cytometry
Uploaded: 2012-02-15T12:00:00
Description: Watch this Scientific Journal Video about Single-cell Analysis of Bacillus subtilis Biofilms Using Fluorescence Microscopy and Flow Cytometry at JoVE.com

Bu çalışma, Würzburg Üniversitesi Enfeksiyon Hastalıkları Araştırma Merkezi (ZINF) gelen, Genç Araştırmacı Araştırma Programı tarafından finanse edilmektedir. Juan C Garcia-Betancur Würzburg Üniversitesi Yaşam Bilimleri Enstitüsü (GSLS) bir doktora üyesidir.

1. Etiketleme B. subtilis ve Biyofilm Oluşumu Deneyi <ol><li> PCR ile, ilgilenilen geni promoter bölgesi amplifiye. Bu örnek olarak P tapa, Tasa matriks proteini 26 üretimi sorumlu genlerin klonlanması arttırıcının göstermektedir. Pkm008 vektör (Özlü laboratuar, Harvard Tıp Okulu. Boston, ABD tarafından yaratılmış) içine Clone P tapa (Şekil 2). </li><li> Enzimatik sindirimi (Enzyme önerilen, XhoI) ile plazmit doğrusallaştırmak. </li><l...

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A., Chu, F., Losick, R., Kolter, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structurally+diverse+natural+products+that+cause+potassium+leakage+trigger+multicellularity+in+Bacillus+subtilis.">Structurally diverse natural products that cause potassium leakage trigger multicellularity in Bacillus subtilis.</a> Proc. Natl. Acad. Sci. U.S.A. 106, 280-285 (2009).</li><li>Lopez, D., Vlamakis, H., Losick, R., Kolter, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cannibalism+enhances+biofilm+development+in+Bacillus+subtilis.">Cannibalism enhances biofilm development in Bacillus subtilis.</a> Mol. 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Bakteri toplulukları genler delillerin mikrobiyal topluluklar 33,34 karmaşıklığı belirli ifade hücrelerin subpopülasyonunun gösteriyor olması. Bu protokol ilgi herhangi bir gen ekspresyonu mikrobiyal toplum içinde özel hücreler belli bir subpopülasyonu kısıtlı olup olmadığını belirlemek için yardımcı olmalıdır. Geleneksel yöntemlerin bütün mikrobiyal topluluk ve mikrobiyal toplum içinde gen ekspresyon dalgalanmalara gen ekspresyonu veya mikroarray analizi oranı gen ekspresyonu d...

<table border="1"><tbody><tr><td> Teknik </td><td> Reaktif Adı </td><td> Şirket </td><td> Katalog numarası </td></tr><tr><td rowspan="11"> Kompozisyon MSgg </td><td> 5 mM potasyum fosfat </td><td> Roth </td><td> 6878 </td></tr><tr><td> 100mM MOPS </td><td> Sigma-Aldrich </td><td> M1254 </td></tr><tr><td> Magnezyum klorür 2mm </td><td> Roth </td><td> 2189.1 </td></tr><tr><td> Kalsiyum klorür 700μM </td><td> Roth </td><td> A119.1 </td></tr><tr><td> Demir klorür 50μM </td><td> Sigma-Aldrich </td><td> 157740 </td></tr><tr><td> Çinko klorid 1 mikrona </td><td> ¸ </td><td> A2076 </td></tr><tr><td> Tiamin 2μM </td><td> Sigma-Aldrich </td><td> 74625 </td></tr><tr><td> Gliserol% 0.5 </td><td> Roth </td><td> 7533 </td></tr><tr><td> Glutadostum% 0.5 </td><td> Sigma-Aldrich </td><td> 49621 </td></tr><tr><td> Triptofan 50μg/ml </td><td> Sigma-Aldrich </td><td> T0254 </td></tr><tr><td> Fenilalanin 50μg/ml </td><td> Sigma-Aldrich </td><td> P2126 </td></tr><tr><td> Hücre fiksasyon </td><td> Paraformaldehyde </td><td> Roth </td><td> 0335 </td></tr><tr><td></td><td> Ekipman Adı </td><td> Şirket </td><td> Katalog numarası </td></tr><tr><td> Sonikasyon </td><td> Hücre Sonicator </td><td> Bandelin </td><td> D-1000 </td></tr><tr><td> Floresans Mikroskopi </td><td> Floresan Mikroskop </td><td> Leica </td><td> DMI6000B </td></tr><tr><td></td><td> Yazılım Adı </td><td> Şirket </td><td> Katalog Numarası </td></tr><tr><td> Floresans Mikroskopi </td> &lt;td&gt; Asaf </td><td> Leica </td><td></td></tr><tr><td> Flow sitometri </td><td> FCASDiva </td><td> BD </td><td></td></tr><tr><td> Flow sitometri </td><td> FlowJo </td><td> Treestar </td><td></td></tr></tbody></table>

single-cell analysis of bacillus subtilis biofilms using fluorescence microscopy and flow cytometry

Biyofilm oluşumu hemen hemen tüm bakterileri 1-6 genel bir niteliktir. Bakteriler biyofilmler oluştururlar, hücreler çoğunlukla 7-10 diğer faktörler arasında proteinler ve ekzopolisakkarit tarafından oluşturulmuştur hücre dışı matris içinde kaplı vardır. Mikrobiyal topluluk genellikle 11-17 özel hücreler farklı subpopülasyonu farklılaşması gösterir biyofilm içinde kaplı. Bu subpopülasyonunun arada ve genellikle 18-21 biyofilm içinde zamansal ve mekansal organizasyonu gösterir. Model organizma Bacillus subtilis Biyofilm oluşumu özel hücreler farklı alt popülasyonlarının farklılaşmayı gerektirir. Bunlar arasında, biyofilm hücre dışı matriks üretmek ve salgılamak sorumlu matriks üreticileri, en subpopülasyonu 11,19 biyofilm oluşumu için gereklidir. Bu nedenle, matris üreticilerinin farklılaşması B. biyofilm oluşumu hallmark subtilis. Biz B. Biyofilmlererde matris üreticilerin subpopülasyonu görselleştirmek ve ölçmek için floresan gazetecilere kullanmış subtilis 15,19,22-24. Somut olarak, bu matris üreticilerinin subpopülasyonu kendi ürettiği ekstraselüler sinyal surfactin 25 varlığında karşılık olarak ayırt olduğu araştırmacılar tarafından gözlenmiştir. İlginç bir şekilde, surfactin matris üreticileri 15 subpopülasyonu farklı özelleşmiş hücre bir alt tarafından üretilir. Bu raporu B. subtilis biyofilmler içinde matrisi üretici ve surfactin üreticilerin subpopülasyonu görselleştirmek ve ölçmek için gerekli teknik yaklaşım ayrıntılı var. Bunu yapmak için, matris üretimi ve surfactin üretimi için gerekli olan genlerin flüoresan gazetecilere B&#39;nin kromozom içine sokulur subtilis. Reporters sadece özel bir hücre subpopülasyonu cinsinden ifade edilmiştir. Daha sonra, alt popülasyonlar olabilirfloresan mikroskobu ve akım sitometri (Şekil 1&#39;e bakınız) kullanılarak izlenir. Özel hücrelerin farklı alt popülasyonlarda bakterilerin çok hücreli topluluklar içinde bir arada olması, bize Prokaryotlarda gen ifadesinin düzenlenmesinde hakkında farklı bir bakış açısı kazandırır. Bu protokol deneysel olarak bu fenomeni adresleri ve kolay bir mikrobiyal topluluk içinde fenotipik heterojenite altında yatan moleküler mekanizmaları aydınlatmak için, başka herhangi bir çalışma modeline adapte edilebilir.

Watch this Scientific Journal Video about Single-cell Analysis of Bacillus subtilis Biofilms Using Fluorescence Microscopy and Flow Cytometry at JoVE.com

Single-cell Analysis of Bacillus subtilis Biofilms Using Fluorescence Microscopy and Flow Cytometry

Mikrobiyal biyofilmler genellikle özel hücreler farklı subpopülasyonunun oluşturduğu edilir. Bu alt popülasyonlarının Tek hücreli analizi floresan gazetecilere kullanımını gerektirir. Burada birkaç subpopulationswithin görselleştirmek ve izlemek için bir protokol tarif<em&gt; B. subtilis</em&gt; Floresan mikroskobu ve akım sitometri kullanarak biyofilm.

Tek hücreli Analizi Bacillus subtilis</emFloresan Mikroskobu ve Akım Sitometri Kullanma> Biyofilmler

Biofilm formation is a general attribute to almost all bacteria 1-6. When bacteria form biofilms, cells are encased in extracellular matrix that is mostly ...

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Immunology and Infection

Single-cell Analysis of Bacillus subtilis Biofilms Using Fluorescence Microscopy and Flow Cytometry

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Live Cell Imaging of Bacillus subtilis and Streptococcus pneumoniae using Automated Time-lapse Microscopy

Live Cell Imaging of Bacillus subtilis and Streptococcus pneumoniae using Automated Time-lapse Microscopy

Canlı Hücre Görüntüleme Bacillus subtilis Ve Streptococcus pneumoniae Otomatik Zaman atlamalı Mikroskopi

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The ability of malignant cells to evade the immune system, characterized by tumor escape from both innate and adaptive immune responses, is now accepted ...

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Isı Önlenmesi ile Sıçanlarda Olumsuz Etkileri Stres<em&gt; Bacillus subtilis</em&gt; Gerilme

This study was designed to evaluate the protective effect of the Bacillus subtilis strain against complications related to heat stress. Thirty-two ...

Methodologies for Studying B. subtilis Biofilms as a Model for Characterizing Small Molecule Biofilm Inhibitors

Methodologies for Studying B. subtilis Biofilms as a Model for Characterizing Small Molecule Biofilm Inhibitors

Araştırma yöntemleri<em&gt; B. subtilis</emKüçük Molekül Biyofilm İnhibitörleri Karakterizasyonu için Bir Model Olarak&gt; Biyofilmler

This work assesses different methodologies to study the impact of small molecule biofilm inhibitors, such as D-amino acids, on the development and ...

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Fare kemik iliğinden plazmasitoid dendritik hücreleri Saflaştırılması floresans ile aktive edilen hücre sınıflandırma

Fluorescence-activated cell sorting (FACS) is a technique to purify specific cell populations based on phenotypes detected by flow cytometry. This method ...

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Yalıtım ve fare ve insan alveoler makrofajlar Vitro kültürü

Alveolar macrophages are terminally differentiated, lung-resident macrophages of prenatal origin. Alveolar macrophages are unique in their long life and ...

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Tek hücreli Immunophenotype ve sitokin üretimde kitle sitometresi ile periferik tam kan analizi

Cytokines play a pivotal role in the pathogenesis of autoimmune diseases. Hence, the measurement of cytokine levels has been the focus of multiple studies ...

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İnsan monosit alt kümeleri tam kan tarafından karakterizasyonu Akış Sitometresi Analizi

Monocytes are key contributors in various inflammatory disorders and alterations to these cells, including their subset proportions and functions, can ...

Quantification of Efferocytosis by Single-cell Fluorescence Microscopy

Efferocytosis tek hücreli floresans mikroskobu tarafından miktar

Studying the regulation of efferocytosis requires methods that are able to accurately quantify the uptake of apoptotic cells and to probe the signaling ...

Tek hücreli Analizi Bacillus subtilis Biyofilmler