Vorremmo ringraziare Ken Nealson per la sua intuizione e consulenza durante tutto il processo di preparazione del manoscritto.

1. Preparazione di sospensioni virali Prima di isolamento dei singoli virioni tramite citometria a flusso, preparare sospensioni virali non fissati. <ol><li> Isolare particelle virali che utilizzano i protocolli stabiliti in precedenza facendo attenzione sospensione virale finale è contenuto in 0,1 micron-filtrata TE (Tris-EDTA, pH 8). Si consiglia di utilizzare filtrazione a flusso tangenziale (TFF) avvicina 6 anche se altri metodi sono stati sviluppati che uso chimico flocculazione 7. </li><li> Enumerare le particelle virali che utilizzano protocolli stabiliti, per esempio usando 8-10 epifluorescenza o citometria a flusso 11,12. </li><li> Aliquota sospensione virale in due provette Eppendorf (volume finale è raccomandato per essere 1.000 ml). </li></ol> 2. Citometria a Flusso <ol><li> Per ordinare i virus utilizzando la BD FACSAria II Citofluorimetro dotato di un PMT diffusione frontale personalizzato (FSC PMT), diluire le particelle virali in 0,1 micron-filtrata TE, pH 8.0 ad un titolo appropriato per un tasso di eventi di 200 eventi sec -1. </li><li> Impostare le soglie per massimizzare razioni segnale-rumore, ad es. per un assemblaggio misto contenente particelle di fago T4 e lambda seguenti sono raccomandati: FSC PMT a 1.000 e SSC a 200. </li><li> Run 100 ml di campioni di &quot;vuoti&quot; che contengono solo 1) 0,1 micron TE-filtrata e 2) 0,1 micron TE-filtrata e SybrGreen1 a 1e-4 diluizione di magazzino (10.000 X). </li><li> Eseguire una piccola aliquota (si consiglia 100 ml) di sospensione virale senza macchia per valutare sfondo, seguita dalla sospensione virale macchiato (SybrGreen1 a 1e-4 diluizione di magazzino), le particelle virali per un totale di 5.000 eventi ciascuno. Questo è per controllare e regolare la concentrazione cancelletti di smistamento, se necessario, prima di ordinamento. Si consiglia un cancello stretto nel centro di particelle virali. Inoltre, per la generazione di cancelli usiamo il SYBR Green e FSC PMT trama. </li><li> Aggiungere 5 ml di 1% basso punto di fusione (LMP) agarosio (in tampone TBE) raffreddata a 37 ° C per ciascunbene su un politetrafluoroetilene (PTFE) vetrino da microscopio (Scienze della microscopia elettronica). </li><li> Caricare il vetrino da microscopio in PTFE nel citofluorimetro per catturare particelle virali ordinati. </li><li> Inizia sorta di particelle virali macchiati SYBR Green direttamente sul PTFE diapositiva con LMP agarosio. Si consiglia di cernita 10 o più eventi (particelle virali) in alcuni pozzi per aiutare nella rilevazione della profondità di virus durante microscopia confocale laser a scansione (CSLM). </li><li> Quando l&#39;ordinamento è completo, rimuovere diapositive dal citofluorimetro e aggiungere 5 ml più LMP agarosio raffreddato a 37 ° C in ogni pozzetto, incorporando così la particella virale (s). </li></ol> 3. Visualizzare Virioni singoli usando la microscopia confocale Se si desidera un singolo virione, quindi determinare se ciascun pozzetto contiene un virus individuo è imperativa e CLSM è necessario per visualizzare il virione incorporato (s) in 3D per verificare che una sola particella è presente e che molti virusnon sono impilati uno sopra l&#39;altro. <ol><li> Impostare il microscopio laser alla lunghezza d&#39;onda di 488 nm di eccitazione. </li><li> Immagine le particelle virali embedded utilizzando un obiettivo 63X lunga distanza di lavoro. </li></ol> 4. Whole Genome Amplification <ol><li> Una volta pozzetti con singoli virus sono identificati con CLSM, eseguire tutta amplificazione del genoma utilizzando più di amplificazione dello spostamento (MDA) nel agarosio (in situ). </li><li> Per diminuire la probabilità di introdurre contaminazioni DNA, rimuovere l&#39;agarosio &#39;perle&#39; desiderate dal vetrino e messo in una provetta sterile PCR. Utilizzare un paio di pinzette sterili e / o lama di rasoio per questo passo. </li><li> Lisare la particella virale in situ utilizzando il calore (94 ° C) per 3 minuti nel blocco termico di un termociclatore. </li><li> Eseguire raccomandazioni MDA reazione seguente del produttore (GenomiPhi kit HY, GE Healthcare), con le seguenti modifiche <ol><li> Ridurre il volume del campione e buf reazionefers a 11,25 e incubare a 30 ° C per un massimo di 2 ore per ridurre l&#39;amplificazione non specifica. </li></ol></li><li> Purificare il materiale genomico amplificato mediante il trasferimento di DNA genomico (gDNA) ad una provetta Eppendorf 1,7 e aggiungere 1/10 del volume 3 M di acetato di sodio (NaOAc) in tampone TBE (stesso tampone utilizzato nella preparazione di agarosio LMP). </li><li> Collocare il campione (s) a 55 ° C per 10 minuti per sciogliere agarosio. </li><li> Spostare tubo (s) a 42 ° C per ridurre la temperatura prima di aggiungere enzima. </li><li> Aggiungere 2 unità β-agarase a ciascuna provetta e incubare per 2 ore. </li><li> Aggiungere 1 volume di una soluzione satura di fenolo tampone al tubo, mescolare vigorosamente e centrifugare a 3500 xg per 15 min. Trasferire il supernatante contenente DNA ad una nuova provetta Eppendorf 1,7 ml. </li><li> Aggiungere un volume di 100% di isopropanolo e 1ml di Glycoblue. Mescolare bene con la metropolitana invertendo. </li><li> Centrifugare a 28.000 xg a 4 ° C per 60 min. Decantare isopropanolo facendo attenzione a non disturbare il pellet. Aggiungere 150 ml di 70% ethaNol ad ogni provetta. Spin a 28.000 xg e RT per 10 min. Decantare l&#39;etanolo, l&#39;aria secca il pellet di DNA e risospendere il DNA in un volume adeguato di tampone TE. </li><li> Si consiglia di ripetere passaggi 4,4-4,9 con le seguenti modifiche. Impostare 3-5 replicano reazioni MDA e ridurre l&#39;incubazione a 30 ° C per 1 ora. Campioni Pool dopo purificazione e precipitato con 95-100% di etanolo per ottenere la concentrazione desiderata. </li></ol> 5. Rappresentante dei risultati La Figura 1 riassume il processo di SVG. Questi metodi utilizzano la citometria a flusso per ordinare particelle virali sul PTFE vetrini contenenti agarosio per isolare singoli virioni da un assemblaggio misto seguito da MDA per ottenere quantità di gDNA che sono sufficienti per il sequenziamento. Come-di-concetto prova, batteriofago lambda e T4 sono stati mescolati e sottoposti al processo SVG 11. Una volta virioni sono stati catturati e inseriti in agarosio, CLSM was usato per visualizzare e confermare che un singolo virione è stato ottenuto;. risultati sono mostrati nella Figura 2 Una volta pozzetti con singoli virioni sono stati identificati, sono stati selezionati per MDA di gDNA. Abbiamo poi utilizzato multiplex PCR specifica per T4 e lambda per identificare il virione isolato, Figura 3. Un isolato fago lambda è stato selezionato per il sequenziamento del genoma. Sequenziamento del genoma è stata effettuata utilizzando la tecnologia 454-titanio e sequenziamento letture sono stati sottoposti a mappatura riferimento per identificare il livello di copertura ottenuta utilizzando SVG. Figura 4 mostra che quasi l&#39;intero genoma lambda stato recuperato (con l&#39;eccezione dei primi 5 bp). <img alt="Figura 1" src="/files/ftp_upload/3899/3899fig1.jpg" /> Figura 1. SVG metodologia. Sospensioni virali contenenti un assemblaggio misto sono ridotti a particelle di virus singoli tramite citometria di flusso che sono poi sorted sui singoli agarosio &quot;perle&quot;. Il virus è inserita all&#39;interno del agarosio tallone sovrapponendo con un ulteriore strato di agarosio messaggio flusso ordinamento citometria. Infine, amplificazione del genoma intero viene eseguita in situ. <img alt="Figura 2" src="/files/ftp_upload/3899/3899fig2.jpg" /> Figura 2. Scansione laser confocale micrografia di una particella virale ordinato incorporato in una perlina di agarosio. A) Ricostruzione tridimensionale di una particella virale SYBR Green I-stained all&#39;interno di una perlina di agarosio. Nel riquadro: un ingrandimento maggiore della particella virale isolato B) Profilo appezzamento di fluorescenza relativa della particella virale singolo macchiato all&#39;interno di un cordone di agarosio.. <img alt="Figura 3" src="/files/ftp_upload/3899/3899fig3.jpg" /> Figura 3. Identificazione batteriofago mediante PCR. A) &lt;/ Strong&gt; Multiplex PCR utilizzando primer T4 e lambda-specifico genotipo, Lanes: 1. TrackIt 1 kb più contatti (Invitrogen), 2. lambda integrasi (750 bp), 3. T4 principale proteina del capside (1.050 bp), 4. . Mix di lambda integrasi e T4 principale proteina del capside B) Dopo la PCR specifico lambda con loci supplementari per confermare ulteriormente fago isolamento genoma, Lanes: 1. Lambda integrasi (750 bp), 2. Lambda repressore (356 bp) 3. Lambda sie (superinfezione esclusione) (456 bp) 4. TrackIt 1 kb più contatti (Invitrogen). <img alt="Figura 4" src="/files/ftp_upload/3899/3899fig4.jpg" /> Figura 4. Lambda attributi del genoma e la copertura a.) GC trama, B) Genoma mappa di lambda (adattato da <a href="http://img.jgi.doe.gov" target="_blank">http://img.jgi.doe.gov</a> ), e C) Mappatura di SVG legge al riferimento batteriofago lambda, asse x è posizione genoma, yaxis è% di copertura.

<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>

Nessun conflitto di interessi dichiarati.

Un certo numero di fattori importanti devono essere prese in considerazione nell&#39;applicazione di approcci SVG. Genotipizzazione, come è stato eseguito durante il proof-of-concept esperimento 13, non è un&#39;opzione valida per gli isolati ambientali o sconosciute come primer conservati non sono disponibili in tutti i gruppi virali. Inoltre, sfondo sintesi del DNA o amplificazione aspecifica è comunemente riportati durante l&#39;amplificazione mediante la reazione di MDA 14. Amplificazione no...

<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'

Tutta l&#39;amplificazione e sequenziamento del genoma delle cellule microbiche singole consente caratterizzazione genomica, senza la necessità di coltivazione 1-3. I virus, che sono onnipresenti e le più numerose entità sul nostro pianeta 4 e importante in tutti gli ambienti 5, devono ancora essere rivelati tramite approcci simili. Qui si descrive un metodo per isolare e caratterizzare il genoma di singoli virioni chiamati &quot;Genomica singolo virus&quot; (SVG). SVG utilizza citometria a flusso per isolare singoli virus e amplificazione del genoma intero di ottenere un&#39;elevata molecolare DNA genomico peso (gDNA) che può essere utilizzato nelle successive reazioni di sequenziamento.

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;

Genomica singolo virus (SVG) è un metodo per isolare e amplificare il genoma di singoli virons. Sospensioni virali di un assemblaggio misto sono ordinati mediante citometria a flusso su un vetrino da microscopio con pozzi discrete contenenti agarosio, catturando così il virione e la riduzione del genoma taglio durante la lavorazione a valle. Intero genoma amplificazione viene ottenuta utilizzando multipla di amplificazione dello spostamento (MDA) risultante in materiale genomico che è adatto per il sequenziamento.

Isolamento e analisi del genoma di virioni singole utilizzando &quot;Genomica singolo 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 Views.  The J. Craig Venter Institute. Genomica singolo virus (SVG) è un metodo per isolare e amplificare il genoma di singoli virons. Sospensioni virali di un assemblaggio misto sono ordinati mediante citometria a flusso su un vetrino da microscopio con pozzi discrete contenenti agarosio, catturando così il virione e la riduzione del genoma taglio durante la lavorazione a valle. Intero genoma amplificazione viene ottenuta utilizzando multipla di amplificazione dello spostamento (MDA) risultante in materiale...

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.

Lo screening RNAi per fattori di accoglienza che partecipano Vaccinia Infezione da virus utilizzando Drosophila Celle

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

Ricerca

Immunologia e infezioni

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.

Separazione di DNA a singolo filamento, doppia elica del DNA e RNA da una Comunità Ambientale virale usando la cromatografia idrossiapatite

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.

Singola cella Analisi Bacillus subtilis I biofilm usando la microscopia a fluorescenza e citometria a flusso

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.

Preparazione di cella singola sospensioni per analisi citofluorimetrica provenienti da diverse regioni della pelle del mouse

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.

Neutrofili Isolamento e analisi per determinare il loro ruolo nel linfoma a cellule sensibilità agli agenti terapeutici

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.

Molto multiplo, la risoluzione super-risoluzione delle celle T utilizzando la madSTORM

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.

Cella singola quantificazione del mRNA e l'espressione di proteine di superficie CD4 infettati da Virus di immunodeficienza delle scimmie+ T cellule isolate da macachi Rhesus

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.

Cella singola analisi di Immunophenotype e produzione di citochine nel sangue periferico intero tramite citometria a 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 di singole comunità batteriche intracellulari generati da un modello murino di infezione delle vie urinarie per analisi unicellulare a valle

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.

Funzionalizzazione dei cantilever a microscopio a forza atomica con cellule A T singole o a particella singola per la spettroscopia immunologica a forza a cella singola

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