Nos gustaría dar las gracias a Ken Nealson por su visión y asesoramiento durante todo el proceso de preparación de manuscritos.

1. Preparación de suspensiones virales Antes de aislamiento de viriones individuales a través de citometría de flujo, preparar suspensiones virales no fijadas. <ol><li> Aislar partículas virales mediante protocolos previamente establecidos que hacen suspensión viral definitiva que figura en 0.1 micras con filtro TE (Tris-EDTA, pH 8). Se recomienda el uso de filtración de flujo tangencial (TFF) se aproxima a 6 aunque otros métodos han sido desarrollados para el uso de químicos de floculación 7. </li><li> Enumerar las partículas virales mediante protocolos establecidos, por ejemplo, utilizando 8-10 epifluorescencia o citometría de flujo de 11,12. </li><li> Alícuota de suspensión viral en dos tubos Eppendorf (volumen final se recomienda tener 1.000 l). </li></ol> 2. Citometría de Flujo <ol><li> Para clasificar los virus utilizando el BD FACSAria II citómetro de flujo equipado con un PMT dispersión frontal personalizado (FSC PMT), diluir las partículas virales en 0,1 micras de filtración TE, pH 8.0 a un título apropiado para una tasa de eventos de 200 eventos seg -1. </li><li> Establecer umbrales para maximizar raciones señal-ruido, por ejemplo. para un conjunto mixto que contiene partículas de fago T4 y lambda se recomiendan las siguientes: FSC PMT en 1000 y SSC a 200. </li><li> Ejecutar 100 l de muestras &quot;en blanco&quot; que sólo contienen 1) 0,1 micras TE-filtrada, y 2) 0,1 micras TE-filtrada y SybrGreen1 a 1e-4 dilución del stock (10.000 X). </li><li> Ejecutar una pequeña alícuota (se recomienda 100 l) de la suspensión viral sin teñir para evaluar fondo, seguido de la suspensión viral manchada (SybrGreen1 a 1e-4 dilución de stock), las partículas virales a un total de 5.000 eventos cada uno. Esto es para comprobar y ajustar la concentración de puertas de clasificación, si es necesario, antes de la especie. Recomendamos una puerta estrecha en el centro de las partículas virales. Además, para la generación de puertas usamos el SYBR Green y FSC PMT trama. </li><li> Añadir 5 l de 1% bajo punto de fusión (LMP) de agarosa (en tampón TBE) se enfrió a 37 ° C a cada unobien en un politetrafluoroetileno (PTFE) portaobjetos de microscopio (Electron Microscopy Sciences). </li><li> Coloque el portaobjetos de PTFE en el citómetro de flujo para capturar las partículas virales clasificadas. </li><li> Comience tipo de partículas virales manchadas SYBR verde directamente en PTFE diapositivas con LMP agarosa. Se recomienda clasificación 10 o más eventos (partículas virales) en algunos pozos para ayudar en la detección de la profundidad de los virus durante la microscopía láser confocal de barrido (CSLM). </li><li> Al ordenar, retire diapositiva del citómetro de flujo y añadir 5 l más LMP agarosa enfriada a 37 º C a cada pocillo, incorporar tanto la partícula viral (s). </li></ol> 3. Visualizar viriones individuales mediante microscopía confocal Si se desea un solo virión, a continuación, determinar si cada pocillo contiene un virus individual es imperativo y CLSM es necesario visualizar el virión incorporado (s) en 3D para verificar que sólo una sola partícula está presente y que múltiples virusno se apilan en la parte superior de uno al otro. <ol><li> Ajuste el microscopio láser a la longitud de onda de 488 nm de excitación. </li><li> Imagen de las partículas virales integrados utilizando un objetivo de larga distancia de trabajo 63X. </li></ol> 4. Amplificación del genoma entero <ol><li> Una vez los pocillos con virus individuales se identifican con CLSM, lleve a cabo la amplificación del genoma entero utilizando amplificación por desplazamiento múltiple (MDA) dentro de la agarosa (in situ). </li><li> Para disminuir la probabilidad de introducir contaminación de ADN, eliminar las &#39;bolas&#39; deseadas de agarosa de la corredera y se coloca en un tubo de PCR estéril. Use un par de pinzas estériles y / o hoja de afeitar para este paso. </li><li> Lyse la partícula viral in situ utilizando el calor (94 ° C) durante 3 min en el bloque de calor de un termociclador. </li><li> Realizar recomendaciones la siguiente reacción MDA del fabricante (kit GenomiPhi HY, GE Healthcare) con las siguientes modificaciones <ol><li> Reducir el volumen de la muestra y la reacción bufcias a 11,25 l y se incuba a 30 ° C durante un máximo de 2 horas para reducir la amplificación no específica. </li></ol></li><li> Se purifica el material genómico amplificado mediante la transferencia de ADN genómico (gDNA) a un tubo Eppendorf de 1,7 ml y añadir 1/10 volúmenes de acetato de sodio 3 M (NaOAc) en tampón TBE (mismo tampón utilizado en la preparación de agarosa LMP). </li><li> Colocar la muestra (s) a 55 ° C durante 10 min para disolver agarosa. </li><li> Mueva tubo (s) a 42 ° C para reducir la temperatura antes de la adición de enzima. </li><li> Añadir 2 unidades de β-agarasa a cada tubo y se incuba durante 2 hr. </li><li> Añadir 1 volumen de una solución de fenol saturado con tampón al tubo, mezclar vigorosamente y se centrifuga a 3500 xg durante 15 min. Transferencia de ADN que contiene sobrenadante a un nuevo tubo Eppendorf de 1,7 ml. </li><li> Añadir un volumen de 100% de isopropanol y 1μl de Glycoblue. Mezclar bien por inversión del tubo. </li><li> Centrifugar a 28.000 xg a 4 ° C durante 60 min. Decantar isopropanol asegurándose de no perturbar el sedimento. Añadir 150 l de 70% ethanol a cada tubo. Girar a 28.000 xg y RT durante 10 min. Decantar el etanol, secar al aire el sedimento de ADN y resuspender el ADN en un volumen apropiado de tampón TE. </li><li> Se recomienda repetir los pasos 4.4 a 4.9 con las siguientes modificaciones. Configurar 3-5 MDA reacciones en paralelo y reducir la incubación a 30 ° C y 1 hora. Piscina muestras después de la purificación y precipitado utilizando etanol 95-100% para obtener la concentración deseada. </li></ol> 5. Los resultados representativos La figura 1 resume el proceso de SVG. Estos métodos utilizan citometría de flujo para clasificar partículas virales en portaobjetos de microscopio de PTFE que contienen agarosa para aislar viriones individuales a partir de un conjunto mezclado seguido por la MDA para obtener cantidades de gDNA que son suficientes para la secuenciación. Como prueba de concepto, bacteriófago lambda y T4 se mezclaron y se sometieron al proceso de SVG 11. Una vez viriones fueron capturados y embebidas en agarosa, CLSM was utiliza para visualizar y confirmar que se obtuvo un único virión;. resultados se muestran en la Figura 2 Una vez que se identificaron los pocillos con viriones individuales, que se seleccionaron para la MDA de ADNg. A continuación, utiliza PCR multiplex específico para T4 y lambda para identificar el virión aislado, Figura 3. Un aislado de fago lambda fue seleccionado para la secuenciación del genoma. La secuenciación del genoma se llevó a cabo utilizando la tecnología de 454-titanio y lecturas de secuenciación fueron sometidos a mapeo de referencia para identificar el nivel de cobertura obtenido usando SVG. Figura 4 muestra que se recuperó casi todo el genoma lambda (con la excepción de los primeros 5 pb). <img alt="Figura 1" src="/files/ftp_upload/3899/3899fig1.jpg" /> Figura 1. SVG metodología. Suspensiones virales que contienen un conjunto mixto se reducen a partículas virales individuales a través de citometría de flujo que luego sorted en individuales agarosa &quot;cuentas&quot;. El virus está incrustado dentro de la perla de agarosa mediante la superposición con una capa adicional de clasificación de citometría de flujo después de agarosa. Por último, todo el genoma de amplificación se lleva a cabo in situ. <img alt="La figura 2" src="/files/ftp_upload/3899/3899fig2.jpg" /> Figura 2. Micrografía de una partícula viral ordenados incrustado en una perla de agarosa de escaneo láser confocal. A) Reconstrucción tridimensional de una partícula viral Verde I-manchado Sybr dentro de una perla de agarosa. Recuadro: un aumento mayor de la partícula viral aislado B) Perfil parcela de fluorescencia relativa de la partícula viral única manchada dentro de un cordón de agarosa.. <img alt="Figura 3" src="/files/ftp_upload/3899/3899fig3.jpg" /> Figura 3. Bacteriófago identificación mediante PCR. A) &lt;/ Strong&gt; Multiplex PCR utilizando primers T4 y lambda-específico para el genotipo, Lanes: 1. TrackIt 1 kb plus escalera (Invitrogen), 2. lambda integrasa (750 pb), 3. Proteína principal de la cápside de T4 (1.050 pb), 4. . Mezcla de lambda integrasa y la principal proteína de la cápside T4 B) Tras lambda específico PCR con loci adicionales para confirmar aún más fago genoma aislamiento, Lanes: 1. Lambda integrasa (750 pb), 2. Represor lambda (356 pb) 3. Lambda sie (exclusión superinfección) (456 pb) 4. TrackIt 1 kb plus escalera (Invitrogen). <img alt="Figura 4" src="/files/ftp_upload/3899/3899fig4.jpg" /> La Figura 4. Lambda atributos del genoma y la cobertura. A) GC trama, B) del mapa del genoma de lambda (adaptado de <a href="http://img.jgi.doe.gov" target="_blank">http://img.jgi.doe.gov</a> ) y C) Asignación de SVG lee en el bacteriófago lambda referencia, eje x es posición del genoma, yaxis es% de 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>

No hay conflictos de interés declarado.

Un número de factores importantes que hay que tener en cuenta a la hora de aplicar enfoques SVG. Genotipificación, como se lleva a cabo durante el experimento de prueba de concepto 13, no es una opción válida para aislamientos ambientales o desconocidos como cebadores conservados no están disponibles en todos los grupos virales. Además, el fondo síntesis de ADN o la amplificación no específica se ha divulgado durante la amplificación mediante la reacción de MDA 14. Amplificación no espe...

<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'

Amplificación del genoma entero y secuenciación de las células microbianas individuales permite la caracterización genómica y sin la necesidad de cultivo de 1-3. Los virus, que son muy abundantes y las más numerosas entidades en nuestro planeta 4 e importante en todos los ambientes, 5 aún no se han puesto de manifiesto a través de enfoques similares. A continuación se describe un método para el aislamiento y la caracterización de los genomas de los viriones single llamado &quot;Genómica Virus única» (SVG). SVG utiliza citometría de flujo para aislar los virus individuales y de todo el genoma de amplificación para obtener ADN genómico de alto peso molecular (gDNA) que se puede utilizar en reacciones de secuenciación posteriores.

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;

Genómica de virus individuales (SVG) es un método para aislar y amplificar los genomas de los viriones individuales. Suspensiones virales de un conjunto mixto se ordenan mediante citometría de flujo en un portaobjetos de microscopio con pozos discretas que contienen agarosa, capturando así el virión y la reducción del genoma cizallamiento durante el procesado posterior. Amplificación del genoma completo se logra mediante la amplificación de desplazamiento múltiple (MDA) resultante en el material genómico que es adecuado para la secuenciación.

Aislamiento y Análisis del Genoma de viriones individuales utilizando &quot;Genómica solo virus &#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 vistas.  The J. Craig Venter Institute. Genómica de virus individuales (SVG) es un método para aislar y amplificar los genomas de los viriones individuales. Suspensiones virales de un conjunto mixto se ordenan mediante citometría de flujo en un portaobjetos de microscopio con pozos discretas que contienen agarosa, capturando así el virión y la reducción del genoma cizallamiento durante el procesado posterior. Amplificación del genoma completo se logra mediante la amplificación de desplazamiento múltipl...

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 detección de los factores del huésped implicados en Vaccinia Infección del virus utilizando Drosophila Celdas

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

Investigación

Inmunología e Infección

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.

La separación de una sola hebra de ADN, el ADN de doble cadena y el ARN de una comunidad ambiental viral mediante cromatografía de 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.

Sola célula de análisis de Bacillus subtilis Las biopelículas mediante microscopía de fluorescencia y citometría de flujo

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.

Visualización de la proteína-DNA en células bacterianas en directo mediante fotoactivado Tracking Single-molécula

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.

Preparación de suspensiones de una sola célula para el Análisis cytofluorimetric de diferentes regiones de piel de ratón

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.

Aislamiento de neutrófilos y análisis para determinar su papel en el linfoma de células sensibles a los agentes terapéuticos

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.

Multiplexado de alta resolución de imágenes de células T utilizando 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.

Análisis unicelular de Immunophenotype y producción de citoquinas en sangre periférica por citometría de masas

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.

Aislamiento de las comunidades bacterianas intracelulares solo generados a partir de un modelo murino de infección del tracto urinario para posteriores análisis unicelular

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.

Funcionalización de voladizos de microscopio de fuerza atómica con células de una t o de una sola partícula para espectroscopia de fuerza de una sola célula inmunológica

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