Biz yazının hazırlık süreci boyunca onun fikir ve tavsiye için Ken Nealson teşekkür etmek istiyorum.

1. Viral süspansiyonlar, bölgesinin hazırlanması Akım sitometri yöntemiyle tek bir virionlar izolasyonu önce, sabitlenmemiş viral süspansiyonlar hazırlamak. <ol><li> Emin yarıyıl sonu viral bir askıya alma yapma daha önce belirlenmiş olan iletişim kuralları&#39;nı kullanarak viral partiküller izole edin 0.1 &#39;de içerdiği almaktadır mikron-süzüldü Yatay aralık TE (Tris-EDTA, pH 8). Diğer yöntemler söz konusu kullanım kimyasal flokülasyon 7 geliştirilmiştir olmasına rağmen Biz yaklaşımlar teğetsel akış filtrasyon (TFF) kullanarak 6 tavsiye ederiz. </li><li> Örneğin Epifloresans 8-10 veya akış sitometri 11,12 kullanarak kurulmuş bir protokolleri, kullanarak viral parçacıkların numaralandırılamıyor. </li><li> Iki Eppendorf tüpleri (son hacim 1.000 ul olması tavsiye edilir) içine kısım viral süspansiyon. </li></ol> 2. Akım Sitometri <ol><li> Özel bir İleri Saçılım PMT (FSC PMT) ile donatılmış BD FACSAria II Akış Sitometresi kullanarak virüsleri sıralamak için, 0.1 yılında viral parçacıkların sulandırmak TE, pH 8 mikron-filtre.0 200 olaylar sn -1 bir olay oranı için uygun bir titresi için. </li><li> Sinyal--gürültü rasyonlarına üst düzeye çıkarmak için eşikleri, örneğin ayarlayın. T4 ve lambda faj parçacıkları aşağıdaki içeren bir karışık malzeme grubuyla karşılaşılmıştır için tavsiye edilir: FSC PMT 1.000 ve SSC az 200 de. </li><li> Sadece 1 içeren &quot;boş&quot; örnekleri) 0,1 mikron-filtre TE ve 2) 1e-4 at 0.1 mikron-filtre TE ve SybrGreen1 stok seyreltme (10.000 X) 100 ul Run. </li><li> Lekeli viral süspansiyon (stokunun 1e-4 dilüsyon at SybrGreen1), 5.000 olaylar her bir toplam için viral parçacıklar tarafından takip arka plan değerlendirmek için boyanmamış viral süspansiyon bir küçük bir kısım (100 ul tavsiye edilir), çalıştırın. Bu tür için önce, gerekirse, konsantrasyon kontrol edin ve sıralama kapıları ayarlamak için olduğunu. Biz viral parçacıkların merkezinde sıkı bir kapısı tavsiye ederiz. Ayrıca, kapılar üreten için biz SYBR Green ve FSC PMT arsa kullanın. </li><li> 1% alçak erime alanına (LMP) ve bölgesinin 5&#39;te ul ekleyin agaroz ile (TBE tamponu otelinin), her ile 37 ° C bulundunuz soğutulduiyi bir politetrafloroetilen (PTFE) mikroskop lamı üzerine (Elektron Mikroskobu Bilimler). </li><li> Sıralama kriteri viral parçacıkların yakalamak için akış sitometresinin içine PTFE mikroskop lamı yükleyin. </li><li> Doğrudan LMP agaroz ile PTFE slayt üzerine SYBR yeşil lekeli viral parçacıkların tür başlayın. Biz konfokal lazer tarama mikroskobu (CSLM) sırasında virüslerin derinliği algılama yardımcı olmak için bazı kuyularda 10 veya daha fazla olaylar (viral parçacıklar) sıralama tavsiye ederiz. </li><li> Sınıflandırıcı tamamlandı olduğunu Ne zaman, akış sitometresinin ışık adlı işletmeye slayt kaldırmak ve yorum eklemek 5 ul devamı LMP, agaroz ve böylece, viral partikül (ler) katıştırma, her bir kuyunun bulundunuz 37 ° C arası ° C&#39;ye soğutuldu. </li></ol> 3. Konfokal Mikroskop kullanarak Tek Kişilik viryonlar görselleştirme , Bir Tek bir virionu arzu edilen edilir halinde, her bir ayrıca bir bireysel virüs içeriyorsa,, o zaman belirlenmesi zorunludur olduğunu ve CLSM yalnızca, bir tek bir parçacık mevcut olduğunu ve bu olduğunu doğrulamak için 3 Boyutlu otelinin katıştırılmış virion (temizle) görselleştirmek için gerekli olan Birden çok virüsHer bir Diğer bölgesinin En iyi ilgili sayısız yığılmış değil, uygulanır. <ol><li> Dalga boyu 488 nm eksitasyon için mikroskop lazer ayarlayın. </li><li> Bir 63X uzun çalışma mesafesi objektif kullanarak katıştırılmış viral parçacıkların Görüntü. </li></ol> 4. Tüm Genom Amplifikasyon <ol><li> Tek virüsleri ile kuyu CLSM ile tanımlanır Bir kez, agaroz (in situ) içinde birden çok yer değiştirme amplifikasyon (MDA) kullanarak tüm genom amplifikasyon gerçekleştirin. </li><li> DNA kontaminasyonu tanıtılması olasılığını azaltmak için, slayt gelen istenen agaroz &#39;boncuk&#39; kaldırmak ve bir steril PCR tüp yerleştirilir. Steril cımbız ve / veya bu adımı için jilet bir çift kullanın. </li><li> In situ bir termalcycler sıcağında bloğunda 3 dk için (94 ° C) ısı kullanılarak en viral parçacık lyse. </li><li> Aşağıdaki değişiklikler ile MDA reaksiyon aşağıdaki üretici tavsiyelerine (GenomiPhi HY kiti, GE Healthcare) gerçekleştirin <ol><li> Örnek ve reaksiyon buf hacmi azaltın30 de 11.25 ul ve inkübe için fers ° nonspesifik amplifikasyon azaltmak için 2 hr bir maksimum için C. </li></ol></li><li> , Bir, 1.7 ml Eppendorf tüp etmek için genomik DNA&#39;sı (gDNA) aktarmak edenler tarafından güçlendirilmiş genomik malzeme Purify ve 1/10 ses seviyesi TBE tamponu otelinin 3 M sodyum asetat (edilerek NaOAc) (aynı tampon maddesi LMP, agaroz hazırlanmasında otelinin ikinci el) ekleyin. </li><li> 55 Place at numune (ler) ° C 10 dakika için agaroz çözmek için. </li><li> 42 için tüp (ler) hareket ettirin ° C enzim eklemeden önce sıcaklığını düşürmek için. </li><li> Her bir tüp ve 2 saat boyunca inkübe edin bulundunuz β-Agarase 2 adet sahipsiniz. </li><li> , Tüp için bir tampon-doymuş bir fenol çözeltisi bölgesinin 1 hacim Ekleyin olarak, şiddetle mix ve 15 dakika Ülke: için 3,500 &#39;den x g&#39;de okuyun santrifüj. , Bir yeni 1.7 ml lik Eppendorf tüp bulundunuz süpernatant ihtiva eden DNA aktarın. </li><li> Glycoblue, bölgesinin% 100, izopropanol ve 1μl bölgesinin bir hacim ekleyin. Tersini tüp tarafından iyi karıştırın. </li><li> 60 dakika için 4 ° C de 28.000 xg&#39;de santrifüjleyin. Durusu izopropanol pelet rahatsız için değil emin yapım. 70% etha 150 ul ekleyinHer bir tüp için nol. 10 dakika için 28.000 xg&#39;de ve oda sıcaklığında Spin. Etanol Durusu için hava, çalıştırıldığında DNA pelet kuru ve TE tampon maddesinde bölgesinin, bir uygun bir hacmi eksiksiz yer alan DNA tekrar süspansiyon haline getirin. </li><li> Biz aşağıdaki modifikasyonlar ile adımları 4,4-4,9 tekrar tavsiye ederiz. Ortalama gönderim süresi 3-5 çoğaltmak MDA reaksiyonları Set up ve 1 saat için 30 de inkübasyon ° C azaltır. Arzu edilen konsantrasyonunun elde edilmesi için% 95-100 aralığı şekilde, etanol kullanılarak ve temizlemesi için çökelti, Aşağıdaki Yüzme Havuzu örnekleri. </li></ol> 5. Temsilcisi Sonuçlar Şekil 1, bu SVG sürecini özetler. Bu yöntemler sıralama için yeterli olan gDNA miktarları elde etmek için MDA tarafından takip bir karışık malzeme grubuyla karşılaşılmıştır gelen tek virionlar izole etmek için agaroz içeren PTFE mikroskop slaytlar üzerine viral parçacıkların sıralamak için akım sitometri kullanın. Bir kanıtı-of-kavram olarak, bakteriyofaj lambda ve T4 karıştırıldı ve SVG Süreci 11 tabi. Virionlar agaroz, CLSM wa yakalanan ve gömülü edildi Bir keztemizle görselleştirmek ve, bir tek bir virionu elde edildi olduğunu teyit etmek için ikinci el;. sonuçları Şekil 2&#39;de otelinin yeri gösterilmiştir arıyorsunuz Bir kez tek kişilik virionlar ile kuyu tespit edilmiştir, onlar gDNA bölgesinin MDA için seçilmişlerdir, edildi. Biz o zaman T4 ve izole virion, Şekil 3. Tanımlamak için lambda Bir izole faj lambda genom sıralama için seçildi için multipleks PCR özel kullanılır. Genom sekanslaması özellikle, ve SVG kullanıyorsanız elde edilen seçenekleri içerisinde bölgesinin seviyeler-ini belirlemek için gerçek zamanlı verilerin referans ilaç politikasına tabi edildi 454-Titanyum teknolojisi ve sıralama okuma kullanılarak gerçekleştirildi. Şekil 4, gösterileri hemen hemen tüm lambda genomu (olanlar önce Herhangi 5&#39;te bp bölgesinin özel durum ile birlikte) kurtarıldı olduğunu. <img alt="Şekil 1" src="/files/ftp_upload/3899/3899fig1.jpg" /> 1 Şekil. Bir karışık malzeme grubuyla karşılaşılmıştır içeren SVG metodolojisi. Viral süspansiyonları daha sonra ler vardır akım sitometri yöntemiyle tek bir virüs parçacıkları indirgenirbireysel agaroz &quot;boncuk&quot; üzerine orted. Virüs agaroz yazılan Mesajı akım sitometrik sıralama ek bir tabaka ile overlaying tarafından agaroz boncuk içinde gömülü edilir. Son olarak, Bütün Genom Dizileme Sistemi amplifikasyon in situ otelinin gerçekleştirilir. <img alt="Şekil 2," src="/files/ftp_upload/3899/3899fig2.jpg" /> Şekil 2. Konfokal lazer bir agaroz boncuk gömülü bir sıralama kriteri viral parçacığın mikrografı tarama. Bir agaroz boncuk içinde bir SYBR Green I-lekeli viral parçacığın A) Üç boyutlu rekonstrüksiyon. Inset: izole viral parçacığın daha yüksek büyütme bir agaroz boncuk içinde lekeli tek bir viral parçacığın göreceli floresans B) Profil arsa.. <img alt="Şekil 3," src="/files/ftp_upload/3899/3899fig3.jpg" /> Şekil 3,. PCR kullanılarak, Bakteriyofaj kimlik belgesi. A) &#39;&lt;/ Strong&gt; Multipleks PCR genotip, Lanes için T4 ve lambda-özel primerler kullanılarak: 1. TrackIt 1 kb artı merdiven (Invitrogen), 2. lambda integraz (750 bp), 3. T4, majör kapsid proteini (1,050 bp), 4. . 1: lambda integraz ve T4 büyük kapsid protein Mix B) ek lokusların ile Bilanço Tarihinden Sonraki lambda spesifik PCR daha fazla faj genom izolasyon, Lanes onaylamak için. Lambda integraz (750 bp), 2. Lambda repressör (356 bp) 3. Lambda sie (süperenfeksiyon hariç tutma) (456 bp) 4. TrackIt 1 kb artı merdiven (Invitrogen). <img alt="Şekil 4," src="/files/ftp_upload/3899/3899fig4.jpg" /> 4 Şekil. Lambda genom nitelikleri ve kapsama. A) GC arsa, B) lambdanın Genom Haritayı (adapte <a href="http://img.jgi.doe.gov" target="_blank">http://img.jgi.doe.gov</a> ), ve C) SVG of Mapping referans bakteriyofaj lambda için okur, x-ekseni olduğu genom konumu, yaxis% kapsama alanı.

<ol>
	<li>Raghunathan, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genomic+DNA+amplification+from+a+single+bacterium.">Genomic DNA amplification from a single bacterium.</a> Appl. Environ. Microbiol. 71, 3342-337 (2005).</li><li>Lasken, R. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Single-cell+genomic+sequencing+using+Multiple+Displacement+Amplification.">Single-cell genomic sequencing using Multiple Displacement Amplification.</a> Curr. Opin. Microbiol. 10, 510-516 (2007).</li><li>Ishoey, T., Woyke, T., Stepanauskas, R., Novotny, M., Lasken, R. 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Çıkar çatışması ilan etti.

SVG yaklaşımlar uygularken önemli bir dizi faktör dikkate alınmalıdır. Genotipleme, proof-of-concept deney 13 sırasında yapılan gibi, korunmuş astarlar tüm viral gruplarında mevcut değildir gibi çevresel veya bilinmeyen izolatların için geçerli bir seçenek değil. Ayrıca, arka plan DNA sentezi ya da spesifik olmayan amplifikasyon sık MDA reaksiyon 14 kullanılarak amplifikasyon sırasında bildirilir. Spesifik olmayan amplifikasyon reaksiyonu reaktif maddeler ve / veya reaksiyo...

<table border="1">
	<tbody>
		<tr>
			<td>Material Name</td>
			<td>Company</td>
			<td>Catalogue Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>1X TE buffer</td>
			<td>Invitrogen</td>
			<td>12090-015</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Buffer-saturated Phenol</td>
			<td>Invitrogen</td>
			<td>15513-039</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>GenomiPhi HY kit</td>
			<td>GE Healthcare</td>
			<td>25-6600-22</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Glycoblue</td>
			<td>Invitrogen</td>
			<td>AM9516</td>
			<td>15 mg/ml</td>
		</tr>
		<tr>
			<td>LMP agarose</td>
			<td>Invitrogen</td>
			<td>16520100</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>PTFE microscope slide</td>
			<td>Electron Microscopy Sciences</td>
			<td>63430-04</td>
			<td>24 well, 4 mm Diameter</td>
		</tr>
		<tr>
			<td>SybrGreen</td>
			<td>Invitrogen</td>
			<td>S7585</td>
			<td>10,000X</td>
		</tr>
		<tr>
			<td>&beta;-agarase</td>
			<td>New England Biolabs</td>
			<td>M0392S</td>
			<td>&nbsp;</td>
		</tr>
	</tbody>
</table>

isolation and genome analysis of single virions using 'single virus genomics'

Tek mikrobiyal hücrelerin tüm genom amplifikasyon ve sıralama ekimi 1-3 en gerek kalmadan genomik karakterizasyonu sağlar. Her yerde ve gezegenimizin 4 üzerindeki en çok sayıda varlıklar ve tüm ortamlarda 5 içinde önemli olan Virüsler,, benzer yaklaşımlar yoluyla ortaya çıkmak için henüz. Burada &#39;Tek Kişilik Virüs Genomik&#39; (SVG) adı verilen tek virionlar genom izole ve karakterize için bir yaklaşım tarif. SVG daha sonraki dizi analizi reaksiyonlar otelinin ikinci el kullanılabilen, yüksek molekül ağırlığı genomik DNA (gDNA) elde etmek üzere tek tek virüs ve Bütün Genom Dizileme Sistemi amplifikasyon izole etmek için akım sitometri kullanır.

Watch this Scientific Journal Video about Isolation and Genome Analysis of Single Virions using 'Single Virus Genomics' at JoVE.com

Isolation and Genome Analysis of Single Virions using &#039;Single Virus Genomics&#039;

Tek Virüs Genomik (SVG) tek Virons en genom izole etmek ve yükseltmek için bir yöntemdir. Karışık malzeme grubuyla viral süspansiyonları böylece virion yakalama ve alt işleme sırasında genom kesme azaltarak, agaroz içeren ayrık kuyu ile bir mikroskop lamı üzerine akım sitometri kullanılarak sıralanır. Tüm genom amplifikasyonu sekanslama için uygun olan genomik malzeme ile sonuçlanan birden fazla yer değiştirme amplifikasyonu (MDA) kullanılarak elde edilir.

&#39;Tek Virüs Genomik&#39; kullanarak Tek viryonlar İzolasyonu ve Genom Analizi

Whole genome amplification and sequencing of single microbial cells enables genomic characterization without the need of cultivation 1-3. Viruses, which ...

isolation-genome-analysis-single-virions-using-single-virus

Research

JoVE Journal

Immunology and Infection

Isolation and Genome Analysis of Single Virions using 'Single Virus Genomics'

10.8K Görüntüleme.  The J. Craig Venter Institute. Tek Virüs Genomik (SVG) tek Virons en genom izole etmek ve yükseltmek için bir yöntemdir. Karışık malzeme grubuyla viral süspansiyonları böylece virion yakalama ve alt işleme sırasında genom kesme azaltarak, agaroz içeren ayrık kuyu ile bir mikroskop lamı üzerine akım sitometri kullanılarak sıralanır. Tüm genom amplifikasyonu sekanslama için uygun olan genomik malzeme ile sonuçlanan birden fazla yer değiştirme amplifikasyonu (MDA) kullanılarak e...

Video: Isolation and Genome Analysis of Single Virions using 'Single Virus Genomics'

RNAi Screening for Host Factors Involved in Vaccinia Virus Infection using Drosophila Cells

Viral pathogens represent a significant public health threat; not only can viruses cause natural epidemics of human disease, but their potential use in bioterrorism is also a concern. A better understanding of the cellular factors that impact infection would facilitate the development of much-needed therapeutics. Recent advances in RNA interference (RNAi) technology coupled with complete genome sequencing of several organisms has led to the optimization of genome-wide, cell-based loss-of-function screens. Drosophila cells are particularly amenable to genome-scale screens because of the ease and efficiency of RNAi in this system 1. Importantly, a wide variety of viruses can infect Drosophila cells, including a number of mammalian viruses of medical and agricultural importance 2,3,4. Previous RNAi screens in Drosophila have identified host factors that are required for various steps in virus infection including entry, translation and RNA replication 5. Moreover, many of the cellular factors required for viral replication in Drosophila cell culture are also limiting in human cells infected with these viruses 4,6,7,8, 9. Therefore, the identification of host factors co-opted during viral infection presents novel targets for antiviral therapeutics. Here we present a generalized protocol for a high-throughput RNAi screen to identify cellular factors involved in viral infection, using vaccinia virus as an example.

RNAi Screening for Host Factors Involved in Vaccinia Virus Infection using Drosophila Cells

Novel host factors involved in viral infection can be identified through cell-based genome-wide loss of function RNAi screening. A Drosophila cell culture model is particularly amenable to this approach due to the ease and efficiency of RNAi. Here we demonstrate this technique using vaccinia virus as an example.

RNAi Tarama için ilgilendim Ana Faktörler Vaccinia Virüs Enfeksiyonu Drosophila Hücre

Viral pathogens represent a significant public health threat; not only can viruses cause natural epidemics of human disease, but their potential use in ...

İmmünoloji ve Enfeksiyon

Separation of Single-stranded DNA, Double-stranded DNA and RNA from an Environmental Viral Community Using Hydroxyapatite Chromatography

Viruses, particularly bacteriophages (phages), are the most numerous biological entities on Earth1,2. Viruses modulate host cell abundance and diversity, contribute to the cycling of nutrients, alter host cell phenotype, and influence the evolution of both host cell and viral communities through the lateral transfer of genes 3. Numerous studies have highlighted the staggering genetic diversity of viruses and their functional potential in a variety of natural environments. 
Metagenomic techniques have been used to study the taxonomic diversity and functional potential of complex viral assemblages whose members contain single-stranded DNA (ssDNA), double-stranded DNA (dsDNA) and RNA genotypes 4-9. Current library construction protocols used to study environmental DNA-containing or RNA-containing viruses require an initial nuclease treatment in order to remove nontargeted templates 10. However, a comprehensive understanding of the collective gene complement of the virus community and virus diversity requires knowledge of all members regardless of genome composition. Fractionation of purified nucleic acid subtypes provides an effective mechanism by which to study viral assemblages without sacrificing a subset of the community&#x2019;s genetic signature. 
Hydroxyapatite, a crystalline form of calcium phosphate, has been employed in the separation of nucleic acids, as well as proteins and microbes, since the 1960s11. By exploiting the charge interaction between the positively-charged Ca2+ ions of the hydroxyapatite and the negatively charged phosphate backbone of the nucleic acid subtypes, it is possible to preferentially elute each nucleic acid subtype independent of the others. We recently employed this strategy to independently fractionate the genomes of ssDNA, dsDNA and RNA-containing viruses in preparation of DNA sequencing 12. Here, we present a method for the fractionation and recovery of ssDNA, dsDNA and RNA viral nucleic acids from mixed viral assemblages using hydroxyapatite chromotography.

We describe an efficient method to separate single-stranded DNA, double-stranded DNA and RNA molecules from environmental viral communities. Nucleic acids are fractionated using hydroxyapatite chromatography with increasing concentrations of phosphate-containing buffers. This method permits the isolation of all viral nucleic acid types from environmental samples.

Tek iplikli DNA, çift iplikli DNA ve RNA Hidroksiapatit Kromatografisi kullanarak bir Çevre Viral Topluluk ayrılması

Viruses, particularly bacteriophages (phages), are the most numerous biological entities on Earth1,2.  Viruses modulate host cell abundance and diversity, ...

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

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 constituted by proteins and exopolysaccharides, among other factors 7-10. The microbial community encased within the biofilm often shows the differentiation of distinct subpopulation of specialized cells 11-17. These subpopulations coexist and often show spatial and temporal organization within the biofilm 18-21.
Biofilm formation in the model organism Bacillus subtilis requires the differentiation of distinct subpopulations of specialized cells. Among them, the subpopulation of matrix producers, responsible to produce and secrete the extracellular matrix of the biofilm is essential for biofilm formation 11,19. Hence, differentiation of matrix producers is a hallmark of biofilm formation in B. subtilis.
We have used fluorescent reporters to visualize and quantify the subpopulation of matrix producers in biofilms of B. subtilis 15,19,22-24. Concretely, we have observed that the subpopulation of matrix producers differentiates in response to the presence of self-produced extracellular signal surfactin 25. Interestingly, surfactin is produced by a subpopulation of specialized cells different from the subpopulation of matrix producers 15.
We have detailed in this report the technical approach necessary to visualize and quantify the subpopulation of matrix producers and surfactin producers within the biofilms of B. subtilis. To do this, fluorescent reporters of genes required for matrix production and surfactin production are inserted into the chromosome of B. subtilis. Reporters are expressed only in a subpopulation of specialized cells. Then, the subpopulations can be monitored using fluorescence microscopy and flow cytometry (See Fig 1).
The fact that different subpopulations of specialized cells coexist within multicellular communities of bacteria gives us a different perspective about the regulation of gene expression in prokaryotes. This protocol addresses this phenomenon experimentally and it can be easily adapted to any other working model, to elucidate the molecular mechanisms underlying phenotypic heterogeneity within a microbial community.

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

Microbial biofilms are generally constituted by distinct subpopulations of specialized cells. Single-cell analysis of these subpopulations requires the use of fluorescent reporters. Here we describe a protocol to visualize and monitor several subpopulationswithin B. subtilis biofilms using fluorescence microscopy and flow cytometry.

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 ...

Preparation of Single-cell Suspensions for Cytofluorimetric Analysis from Different Mouse Skin Regions

The skin is a barrier organ that interacts with the external environment. Being continuously exposed to potential microbial invasion, the dermis and epidermis home a variety of immune cells in both homeostatic and inflammatory conditions. Tools to obtain skin cell release for cytofluorimetric analyses are, therefore, very useful in order to study the complex network of immune cells residing in the skin and their response to microbial stimuli. Here, we describe an efficient methodology for the digestion of mouse skin to rapidly and efficiently obtain single-cell suspensions. This protocol allows maintenance of maximum cell viability without compromising surface antigen expression. We also describe how to take and digest skin samples from different anatomical locations, such as the ear, trunk, tail, and footpad. The obtained suspensions are then stained and analyzed by flow cytometry to discriminate between different leukocyte populations.

The skin is home to a complex immune cell network. We describe an efficient methodology for the digestion of mouse skin, from different parts of the animal's body, in order to obtain a single-cell suspension and analyze the different leukocyte populations resident in the skin by flow cytometry.

Farklı Fare Cilt Bölgelerinden sitoflüorimetrik analize için tek hücre süspansiyonları hazırlanması

The skin is a barrier organ that interacts with the external environment. Being continuously exposed to potential microbial invasion, the dermis and ...

Neutrophil Isolation and Analysis to Determine their Role in Lymphoma Cell Sensitivity to Therapeutic Agents

Neutrophils are the most abundant (40% to 75%) type of white blood cells and among the first inflammatory cells to migrate towards the site of inflammation. They are key players in the innate immune system and play major roles in cancer biology. Neutrophils have been proposed as key mediators of malignant transformation, tumor progression, angiogenesis and in the modulation of the antitumor immunity; through their release of soluble factors or their interaction with tumor cells. To characterize the specific functions of neutrophils, a fast and reliable method is coveted for in vitro isolation of neutrophils from human blood. Here, a density gradient separation method is demonstrated to isolate neutrophils as well as mononuclear cells from the blood. The procedure consists of layering the density gradient solution such as Ficoll carefully above the diluted blood obtained from patients diagnosed with chronic lymphocytic leukemia (CLL), followed by centrifugation, isolation of mononuclear layer, separation of neutrophils from RBCsby dextran then lysis of residual erythrocytes. This method has been shown to isolate neutrophils &#8805; 90 % pure. To mimic the tumor microenvironment, 3-dimensional (3D) experiments were performed using basement membrane matrix such as Matrigel. Given the short half-life of neutrophils in vitro, 3D experiments with fresh human neutrophils cannot be performed. For this reason promyelocytic HL60 cells are differentiated along the granulocytic pathway using the differentiation inducers dimethyl sulfoxide (DMSO) and retinoic acid (RA). The aim of our experiments is to study the role of neutrophils on the sensitivity of lymphoma cells to anti-lymphoma agents. However these methods can be generalized to study the interactions of neutrophils or neutrophil-like cells with a large range of cell types in different situations.

Neutrophils are the most abundant type of white blood cells. The isolation of neutrophils from human blood by density gradient separation method and the differentiation of human promyelocytic (HL60) cells along the granulocytic pathway are described here; to test their role on sensitivity of lymphoma cells to anti-lymphoma agents.

Nötrofil İzolasyon ve Analiz Terapötik Ajanların için Lenfoma Hücre Duyarlılık kendi Rolü belirleme

Neutrophils are the most abundant (40% to 75%) type of white blood cells and among the first inflammatory cells to migrate towards the site of ...

Highly Multiplexed, Super-resolution Imaging of T Cells Using madSTORM

Imaging heterogeneous cellular structures using single molecule localization microscopy has been hindered by inadequate localization precision and multiplexing ability. Using fluorescent nano-diamond fiducial markers, we describe the drift correction and alignment procedures required to obtain high precision in single molecule localization microscopy. In addition, a new multiplexing strategy, madSTORM, is described in which multiple molecules are targeted in the same cell using sequential binding and elution of fluorescent antibodies. madSTORM is demonstrated on an activated T cell to visualize the locations of different components within a membrane-bound, multi-protein structure called the T cell receptor microcluster. In addition, application of madSTORM as a general tool for visualization of multi-protein structures is discussed.

We demonstrate a method to image multiple molecules within heterogeneous nano-structures at single molecule accuracy using sequential binding and elution of fluorescently labeled antibodies.

Madstorm kullanarak T hücrelerinin çok yönlü, süper çözünürlüklü görüntüsü

Imaging heterogeneous cellular structures using single molecule localization microscopy has been hindered by inadequate localization precision and ...

Single-cell Quantitation of mRNA and Surface Protein Expression in Simian Immunodeficiency Virus-infected CD4+ T Cells Isolated from Rhesus macaques

Single-cell analysis is an important tool for dissecting heterogeneous populations of cells. The identification and isolation of rare cells can be difficult. To overcome this challenge, a methodology combining indexed flow cytometry and high-throughput multiplexed quantitative polymerase chain reaction (qPCR) was developed. The objective was to identify and characterize simian immunodeficiency virus (SIV)-infected cells present within rhesus macaques. Through quantitation of surface protein by fluorescence-activated cell sorting (FACS) and mRNA by qPCR, virus-infected cells are identified by viral gene expression, which is combined with host gene and protein measurements to create a multidimensional profile. We term the approach, targeted Single-Cell Proteo-transcriptional Evaluation, or tSCEPTRE. To perform the method, viable cells are stained with fluorescent antibodies specific for surface markers used for FACS isolation of a cell subset and/or downstream phenotypic analysis. Single cells are sorted followed by immediate lysis, multiplex reverse transcription (RT), PCR pre-amplification, and high throughput qPCR of up to 96 transcripts. FACS measurements are recorded at the time of sorting and subsequently linked to the gene expression data by well position to create a combined protein and transcriptional profile. To study SIV-infected cells directly ex vivo, cells were identified by qPCR detection of multiple viral RNA species. The combination of viral transcripts and the quantity of each provide a framework for classifying cells into distinct stages of the viral life cycle (e.g., productive versus non-productive). Moreover, tSCEPTRE of SIV+ cells were compared to uninfected cells isolated from the same specimen to assess differentially expressed host genes and proteins. The analysis revealed previously unappreciated viral RNA expression heterogeneity among infected cells as well as in vivo SIV-mediated post-transcriptional gene regulation with single-cell resolution. The tSCEPTRE method is relevant for the analysis of any cell population amenable to identification by expression of surface protein marker(s), host or pathogen gene(s), or combinations thereof.

Single-cell Quantitation of mRNA and Surface Protein Expression in Simian Immunodeficiency Virus-infected CD4+ T Cells Isolated from Rhesus macaques

Described is a methodology to quantitate the expression of 96 genes and 18 surface proteins by single cells ex vivo, allowing for the identification of differentially expressed genes and proteins in virus-infected cells relative to uninfected cells. We apply the approach to study SIV-infected CD4+ T cells isolated from rhesus macaques.

Tek hücreli Nefelometri mRNA ve yüzey Protein Simian virüs, immün yetmezlik enfekte CD4 ifadede+ T hücreleri izole Rhesus makak üzerinden

Single-cell analysis is an important tool for dissecting heterogeneous populations of cells. The identification and isolation of rare cells can be ...

Single-cell Analysis of Immunophenotype and Cytokine Production in Peripheral Whole Blood via Mass Cytometry

Cytokines play a pivotal role in the pathogenesis of autoimmune diseases. Hence, the measurement of cytokine levels has been the focus of multiple studies in an attempt to understand the precise mechanisms that lead to the breakdown of self-tolerance and subsequent autoimmunity. Approaches thus far have been based on the study of one specific aspect of the immune system (a single or few cell types or cytokines), and do not offer a global assessment of complex autoimmune disease. While patient sera-based studies have afforded important insights into autoimmunity, they do not provide the specific cellular source of the dysregulated cytokines detected. A comprehensive single-cell approach to evaluate cytokine production in multiple immune cell subsets, within the context of &#34;intrinsic&#34; patient-specific plasma circulating factors, is described here. This approach enables monitoring of the patient-specific immune phenotype (surface markers) and function (cytokines), either in its native &#34;intrinsic pathogenic&#34; disease state, or in the presence of therapeutic agents (in vivo or ex vivo).

Here we describe a single-cell proteomic approach to evaluate immune phenotypic and functional (intracellular cytokine induction) alterations in peripheral whole blood samples, analyzed via mass cytometry.

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 ...

Isolation of Single Intracellular Bacterial Communities Generated from a Murine Model of Urinary Tract Infection for Downstream Single-cell Analysis

In this article, we outline a procedure used to isolate individual intracellular bacterial communities from a mouse that has been experimentally infected in the urinary tract. The protocol can be broadly divided into three sections: the infection, bladder epithelial cell harvesting, and mouth micropipetting to isolate individual infected epithelial cells. The isolated epithelial cell contains viable bacterial cells and is nearly free of contaminating extracellular bacteria, making it ideal for downstream single-cell analysis. The time taken from the start of infection to obtaining a single intracellular bacterial community is about 8 h. This protocol is inexpensive to deploy and uses widely available materials, and we anticipate that it can also be utilized in other infection models to isolate single infected cells from cell mixtures even if those infected cells are rare. However, due to a potential risk in mouth micropipetting, this procedure is not recommended for highly infectious agents.

This protocol describes a simple method of isolating single, infected-bladder epithelial cells from a murine model of urinary tract infection.

İdrar yolu enfeksiyonu aşağı akım tek hücreli analiz için bir fare modelinden oluşturulan tek hücre içi bakteriyel toplulukların yalıtım

In this article, we outline a procedure used to isolate individual intracellular bacterial communities from a mouse that has been experimentally infected ...

Functionalization of Atomic Force Microscope Cantilevers with Single-T Cells or Single-Particle for Immunological Single-Cell Force Spectroscopy

Atomic force microscopy based single cell force spectroscopy (AFM-SCFS) is a powerful tool for studying biophysical properties of living cells. This technique allows for probing interaction strengths and dynamics on a live cell membrane, including those between cells, receptor and ligands, and alongside many other variations. It also works as a mechanism to deliver a physical or biochemical stimulus on single cells in a spatiotemporally controlled manner, thus allowing specific cell activation and subsequent cellular events to be monitored in real-time when combined with live-cell fluorescence imaging. The key step in those AFM-SCFS measurements is AFM-cantilever functionalization, or in other words, attaching a subject of interest to the cantilever. Here, we present methods to modify AFM cantilevers with a single T cell and a single polystyrene bead respectively for immunological studies. The former involves a biocompatible glue that couples single T cells to the tip of a flat cantilever in a solution, while the latter relies on an epoxy glue for single bead adhesion in the air environment. Two immunological applications associated with each cantilever modification are provided as well. The methods described here can be easily adapted to different cell types and solid particles.

We present a protocol to functionalize atomic force microscope (AFM) cantilevers with a single T cell and bead particle for immunological studies. Procedures to probe single-pair T cell-dendritic cell binding by AFM and to monitor the real-time cellular response of macrophages to a single solid particle by AFM with fluorescence imaging are shown.

Tek-T hücreleri veya tek parçacık Immünolojik tek hücreli kuvvet spektroskopisi ile Atomic Force mikroskop Cantilevers functionalization

Atomic force microscopy based single cell force spectroscopy (AFM-SCFS) is a powerful tool for studying biophysical properties of living cells. This ...