Gostaríamos de agradecer a Ken Nealson por sua visão e aconselhamento ao longo do processo de preparação do manuscrito.

1. Preparação de suspensões virais Antes de isolamento do vírus da única via citometria de fluxo, preparar suspensões virais não corrigidos. <ol><li> Isolar as partículas virais utilizando protocolos previamente estabelecidos, tornando-se a suspensão virai final, está contida em 0,1 micrometros de filtrado de TE (Tris-EDTA, pH 8). Recomendamos o uso de filtração de fluxo tangencial (TFF) se aproxima 6 embora outros métodos foram desenvolvidos que uso de produtos químicos de floculação 7. </li><li> Enumerar partículas virais utilizando protocolos estabelecidos, por exemplo, usando 8-10 epifluorescência ou citometria de fluxo 11,12. </li><li> Alíquotas de suspensão viral em dois tubos Eppendorf (volume final é recomendado para ser 1000 ul). </li></ol> 2. Citometria de Fluxo <ol><li> Para classificar os vírus usando o BD FACSAria II citômetro de fluxo equipado com um PMT Forward Scatter personalizado (FSC PMT), diluir partículas virais em 0,1 mM-filtrada TE, pH 8.0 com um título adequado para uma taxa de eventos de 200 eventos seg -1. </li><li> Definir limites para maximizar rações sinal-ruído, por exemplo. para uma assembléia mista contendo partículas de fago T4 e lambda se recomenda o seguinte: FSC PMT em 1000 e SSC em 200. </li><li> Executar 100 ml de amostras &quot;em branco&quot;, contendo apenas 1) TE 0,1 mM-filtrada e 2) TE 0,1 mM-filtrada e SybrGreen1 em 1e-4 diluição de estoque (10.000 X). </li><li> Executar uma pequena aliquota (100 uL é recomendado) de suspensão viral não coradas para avaliar a fundo, seguido por suspensão viral coradas (SybrGreen1 a 1e-4 de diluição de estoque), as partículas virais num total de 5.000 eventos cada. Isto é para verificar e ajustar a concentração de triagem portas, se necessário, antes da classificação. Recomenda-se uma porta estanque no centro de partículas virais. Além disso, para a geração de portas usamos o verde SYBR e FSC PMT trama. </li><li> Adicionar 5 uL de 1% de baixo ponto de fusão (LMP) de agarose (em tamp TBE), arrefecida a 37 ° C para cadabem em um politetrafluoretileno (PTFE) lâmina de microscópio (Electron Microscopy Sciences). </li><li> Carregar a lâmina de microscópio de PTFE no citómetro de fluxo para capturar classificados partículas virais. </li><li> Comece tipo de SYBR Green manchadas de partículas virais diretamente em PTFE de slides com LMP agarose. Recomendamos triagem 10 ou mais acontecimentos (partículas virais) em alguns poços para auxiliar na detecção da profundidade de vírus durante a microscopia confocal laser scanning (CSLM). </li><li> Quando a separação está completa, remover a lâmina do citómetro de fluxo e adicionam-se 5 ul de agarose LMP mais arrefecida até 37 ° C a cada poço, incorporando assim a partícula virai (s). </li></ol> 3. Visualizando Virions única usando microscopia confocal Se uma única virião é desejado, em seguida, determinar se cada poço contém um vírus indivíduo é imperativo e MCVL é necessário visualizar o virião incorporado (s) em 3D para verificar que apenas uma única partícula está presente e que vários vírusnão são empilhadas em cima umas das outras. <ol><li> Definir o microscópio de laser no comprimento de onda de 488 nm de excitação. </li><li> Imagem das partículas virais incorporadas usando um longo objetiva 63X distância de trabalho. </li></ol> 4. Amplificação do genoma inteiro <ol><li> Depois de os poços com vírus individuais são identificados com CLSM, realizar amplificação do genoma completo, utilizando amplificação por deslocamento múltipla (MDA) na agarose (in situ). </li><li> Para diminuir o risco de introdução de contaminação de DNA, remover os desejados &#39;&#39; grânulos de agarose a corrediça e colocado num tubo estéril de PCR. Utilizar um par de pinças esterilizadas e / ou lâmina de barbear para esta etapa. </li><li> Lisar as partículas virais in situ, utilizando calor (94 ° C) durante 3 min no bloco de calor de um termociclador. </li><li> Realizar recomendações reação do fabricante seguinte, o MDA (GenomiPhi HY kit, GE Healthcare) com as seguintes modificações <ol><li> Reduzir o volume de amostra e de reacção bufrências para 11,25 mL e incubar a 30 ° C por um período máximo de 2 horas para reduzir a amplificação inespecífica. </li></ol></li><li> Purifica-se o material genómico amplificado por transferência de ADN genómico (gDNA) para um tubo Eppendorf de 1,7 ml e adicione 1/10 do volume de 3 M de acetato de sódio (NaOAc) em tampão TBE (o mesmo tampão usado na preparação de agarose LMP). </li><li> Colocar a amostra (s), a 55 ° C durante 10 minutos para dissolver agarose. </li><li> Mover tubo (s) a 42 ° C para reduzir a temperatura antes da adição da enzima. </li><li> Adicionar duas unidades β-agarase a cada tubo e incubar durante 2 horas. </li><li> Adicionar 1 volume de uma solução de fenol saturado com tampão ao tubo, misturar vigorosamente e centrifugar a 3500 x g durante 15 min. Transferir o sobrenadante contendo o ADN para um novo tubo Eppendorf de 1,7 ml. </li><li> Adicionar um volume de 100% de isopropanol e 1 ul de Glycoblue. Misture bem, invertendo o tubo. </li><li> Centrifugar a 28.000 xg, a 4 ° C durante 60 min. Decantar isopropanol tomando cuidado para não perturbar o sedimento. Adicionar 150 ul de 70% ethanol a cada tubo. Girar a 28.000 xg e RT durante 10 min. Decantar o etanol, o ar seco do sedimento de DNA e ressuspender o ADN num volume apropriado de tampão TE. </li><li> Recomendamos repetindo os passos 4,4-4,9 com as seguintes modificações. Configurar 3-5 replicados reacções MDA e reduzir a incubação a 30 ° C e 1 hora. Piscina amostras após purificação e precipitado com etanol a 95-100% para se obter a concentração desejada. </li></ol> 5. Os resultados representativos A Figura 1 resume o processo SVG. Esses métodos usam a citometria de fluxo para classificar partículas virais em PTFE lâminas de microscópio contendo agarose para isolar virions único a partir de um conjunto misto, seguido pelo MDA para obter quantidades de gDNA que são suficientes para o seqüenciamento. Como uma prova de conceito, bacteriófago lambda e T4 foram misturados e submetidos ao processo de SVG 11. Uma vez que os viriões foram capturadas e embebidas em agarose, MCVL was utilizados para visualizar e confirmar que um único virião foi obtido,. resultados são mostrados na Figura 2 Uma vez que os poços com viriões individuais foram identificados, que foram seleccionados para MDA de gDNA. Em seguida, utilizado a PCR multiplex específico para T4 e lambda para identificar o virião isolado, Figura 3. Um isolado do fago lambda foi seleccionado para sequenciação do genoma. A sequenciação do genoma foi realizada utilizando a tecnologia 454-titânio e sequenciação leituras foram submetidos a mapeamento de referência para identificar o grau de cobertura obtida usando SVG. Figura 4 mostra que a quase totalidade do genoma de lambda foi recuperado (com a excepção dos primeiros 5 bp). <img alt="Figura 1" src="/files/ftp_upload/3899/3899fig1.jpg" /> Figura 1. SVG metodologia. Suspensões virais contendo uma assembléia mista são reduzidos a partículas do vírus individuais através de citometria de fluxo, que são, em seguida, sorted para cada agarose &quot;beads&quot;. O vírus está incorporado no agarose talão sobrepondo com uma camada adicional de agarose pós classificação por citometria de fluxo. Por último, a amplificação do genoma inteiro é realizada in situ. <img alt="Figura 2" src="/files/ftp_upload/3899/3899fig2.jpg" /> Figura 2. Micrografia de varrimento de laser confocal de uma partícula viral classificados incorporado num grânulo de agarose a.) Reconstrução tridimensional de SYBR Green I-manchado partícula viral dentro de um grânulo de agarose. Detalhe: maior ampliação da partícula viral isolado B) lote de fluorescência relativa do manchado partícula viral único dentro de um talão de agarose Perfil.. <img alt="Figura 3" src="/files/ftp_upload/3899/3899fig3.jpg" /> Figura 3. Identificação bacteriófago utilizando PCR. D) &lt;/ Strong&gt; Multiplex PCR usando primers T4 e específico lambda para genótipo, Lanes: 1. TrackIt 1 kb, mais escada (Invitrogen), 2. lambda integrase (750 pb), 3. Principal proteína da cápside T4 (1050 bp), 4. . Mix de lambda integrase e T4 principal proteína da cápside B) Após lambda específico PCR com loci adicionais para confirmar ainda mais o isolamento do genoma de fagos, Lanes: 1. Lambda integrase (750 pb), 2. Repressor Lambda (356 bp) 3. Lambda sie (exclusão superinfecção) (456 pb) 4. TrackIt 1 kb além de escada (Invitrogen). <img alt="Figura 4" src="/files/ftp_upload/3899/3899fig4.jpg" /> Figura 4. Lambda genoma atributos e cobertura a.) GC enredo, B) Genome mapa de lambda (adaptado de <a href="http://img.jgi.doe.gov" target="_blank">http://img.jgi.doe.gov</a> ) e C) Mapeamento de SVG lê a referência bacteriófago lambda, x-eixo é posição do genoma, yaxis é a cobertura%.

<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. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genomic+sequencing+of+single+microbial+cells+from+environmental+samples.">Genomic sequencing of single microbial cells from environmental samples.</a> Curr. Opin. Microbiol. 11, 198-204 (2008).</li><li>Edwards, R. A., Rohwer, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Viral+metagenomics.">Viral metagenomics.</a> Nature Reviews Microbiology. 3, 504-510 (2005).</li><li>Rohwer, F., Prangishvili, D., Lindell, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Roles+of+viruses+in+the+environment.">Roles of viruses in the environment.</a> Environ. Microbiol. 11, 2771-2774 (2009).</li><li>Williamson, S. J., 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=The+Sorcerer+II+Global+Ocean+Sampling+Expedition:+metagenomic+characterization+of+viruses+within+aquatic+microbial+samples.">The Sorcerer II Global Ocean Sampling Expedition: metagenomic characterization of viruses within aquatic microbial samples.</a> PLoS ONE. 3, e1456 (2008).</li><li>John, S. G., 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=A+simple+and+efficient+method+for+concentration+of+ocean+viruses+by+chemical+flocculation.">A simple and efficient method for concentration of ocean viruses by chemical flocculation.</a> Environ. Microbiol. Rep. 3, 195-202 (2011).</li><li>Noble, R., Fuhrman, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Use+of+SYBR+Green+I+for+rapid+epifluorescence+counts+of+marine+viruses+and+bacteria.">Use of SYBR Green I for rapid epifluorescence counts of marine viruses and bacteria.</a> Aquat Microb Ecol. 14, 113-118 (1998).</li><li>Hara, S., Terauchi, K., Koike, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Abundance+of+viruses+in+marine+waters:+assessment+by+epifluorescence+and+transmission+electron+microscopy.">Abundance of viruses in marine waters: assessment by epifluorescence and transmission electron microscopy.</a> Appl. Environ. Microbiol. 57, 2731-2734 (1991).</li><li>Hennes, K. P., Suttle, C. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Direct+counts+of+viruses+in+natural+waters+and+laboratory+cultures+by+epifluorescence+microscopy.">Direct counts of viruses in natural waters and laboratory cultures by epifluorescence microscopy.</a> Limnol. Oceanogr. 40, 1050-1055 (1995).</li><li>Marie, D., Brussaard, C. P. D., Thyrhaug, R., Bratbak, G., Vaulot, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Enumeration+of+marine+viruses+in+culture+and+natural+samples+by+flow+cytometry.">Enumeration of marine viruses in culture and natural samples by flow cytometry.</a> Appl. Environ. Microbiol. 65, (1999).</li><li>Brussaard, C. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Enumeration+of+bacteriophages+using+flow+cytometry.">Enumeration of bacteriophages using flow cytometry.</a> Methods Mol. Biol. 501, 97-111 (2009).</li><li>Allen, L. Z., 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=Single+Virus+Genomics:+A+New+Tool+for+Virus+Discovery.">Single Virus Genomics: A New Tool for Virus Discovery.</a> Plos One. 6, (2011).</li><li>Hutchison, C. A., Smith, H. O., Pfannkoch, C., Venter, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell-free+cloning+using+phi+29+DNA+polymerase.">Cell-free cloning using phi 29 DNA polymerase.</a> Proceedings of the National Academy of Sciences of the United States of America. 102, 17332-17336 (2005).</li></ol>

Não há conflitos de interesse declarados.

Uma série de fatores importantes devem ser levados em consideração na aplicação de abordagens SVG. Genotipagem, que foi realizada durante o experimento prova-de-conceito 13, não é uma opção válida para os isolados ambientais ou desconhecidos como iniciadores conservados não estão disponíveis em todos os grupos virais. Além disso, a síntese de ADN de fundo, ou a amplificação não específica é comumente avaliado durante a amplificação usando a reacção do MDA 14. Amplificação ...

<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'

Amplificação e seqüenciamento do genoma inteiro das células microbianas individuais permite a caracterização genômica, sem a necessidade de cultivo 1-3. Os vírus, que são onipresentes e as mais numerosas entidades em nosso planeta 4 e importante em todos os ambientes 5, têm ainda a ser revelado através de abordagens semelhantes. Aqui nós descrevemos um método para isolar e caracterizar os genomas de virions single chamado &#39;Genomics Vírus único »(SVG). SVG utiliza a citometria de fluxo para isolar os vírus de amplificação individuais e todo o genoma para obter ADN genómico de elevado peso molecular (gDNA), que pode ser utilizado em reacções de sequenciação subsequentes.

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;

Genomics vírus individuais (SVG) é um método para isolar e amplificar os genomas de virons individuais. Suspensões virais de uma assembléia mista são classificados usando citometria de fluxo em uma lâmina de microscópio com poços discretos contendo agarose, capturando assim, o virion e reduzindo corte genoma durante o processamento downstream. Amplificação do genoma inteiro é conseguida usando vários amplificação por deslocamento (MDA), resultando em material genómico que é adequado para a sequenciação.

Isolamento e Análise do Genoma do vírus da única usando &#39;Genomics vírus simples&#39;

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 visualizações.  The J. Craig Venter Institute. Genomics vírus individuais (SVG) é um método para isolar e amplificar os genomas de virons individuais. Suspensões virais de uma assembléia mista são classificados usando citometria de fluxo em uma lâmina de microscópio com poços discretos contendo agarose, capturando assim, o virion e reduzindo corte genoma durante o processamento downstream. Amplificação do genoma inteiro é conseguida usando vários amplificação por deslocamento (MDA), resultando em materi...

Vídeo: 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 Triagem para Fatores Hospedeiros Envolvidos na Vaccinia Infecção pelo Vírus usando Drosophila Células

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

Imunologia e Infecção

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.

Separação de Single-stranded DNA, Double-stranded DNA e RNA de uma Comunidade Ambiental Viral Usando Cromatografia hidroxiapatita

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.

Análise de uma única célula de Bacillus subtilis Os biofilmes utilizando microscopia de fluorescência e citometria de fluxo

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 ...

Visualizing Protein-DNA Interactions in Live Bacterial Cells Using Photoactivated Single-molecule Tracking

Protein-DNA interactions are at the heart of many fundamental cellular processes. For example, DNA replication, transcription, repair, and chromosome organization are governed by DNA-binding proteins that recognize specific DNA structures or sequences. In vitro experiments have helped to generate detailed models for the function of many types of DNA-binding proteins, yet, the exact mechanisms of these processes and their organization in the complex environment of the living cell remain far less understood. We recently introduced a method for quantifying DNA-repair activities in live Escherichia coli cells using Photoactivated Localization Microscopy (PALM) combined with single-molecule tracking. Our general approach identifies individual DNA-binding events by the change in the mobility of a single protein upon association with the chromosome. The fraction of bound molecules provides a direct quantitative measure for the protein activity and abundance of substrates or binding sites at the single-cell level. Here, we describe the concept of the method and demonstrate sample preparation, data acquisition, and data analysis procedures.

Photoactivated localization microscopy (PALM) combined with single-molecule tracking allows direct observation and quantification of protein-DNA interactions in live Escherichia coli cells.

Visualizando interações DNA-proteína em células bacterianas ao vivo usando fotoativado única molécula de Rastreamento

Protein-DNA interactions are at the heart of many fundamental cellular processes. For example, DNA replication, transcription, repair, and chromosome ...

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.

Preparação de uma única célula suspensões para Análise de citometria de fluxo de diferentes regiões da pele do rato

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.

Isolamento de neutrófilos e análise para determinar seu papel na linfoma sensibilidade das células a agentes terapêuticos

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.

Imagem altamente multiplexada e de super resolução de células T usando madSTORM

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

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.

Análise de célula única do imunofenótipo e produção de citocinas no sangue periférico através de citometria de massa

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.

Isolamento de comunidades de bacterianas intracelulares único gerado a partir de um modelo murino de infecção do trato urinário para análise de célula única a jusante

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.

Functionalization dos Cantialavancos do microscópio da força atômica com únicas-células de T ou único-partícula para a espectroscopia imunológica da força da único-pilha

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