Questo lavoro è stato finanziato dal Medical Consiglio Nazionale delle Ricerche, Singapore.

1. Alexa Fluor etichettatura dei virus dengue <ol><li> Prima che la reazione di marcatura, purificare virus della dengue con cuscino di saccarosio e preparare i reagenti necessari e le attrezzature, come indicato nel protocollo. </li><li> Preparare fresca 0,2 M tampone bicarbonato di sodio, pH 8,5 (tampone etichettatura), e 1,5 M tampone idrossilammina, pH 8.3 (fermata reagente), poco prima di etichettatura e filtro sterilizzare con filtri per siringa 0.2μm. </li><li> Diluire a circa 3x10 8 unità placca formando (PFU) di depurata virus della dengue in 1 ml di etichettatura tampone in una provetta 2 ml. Questo può essere scalata proporzionalmente per l&#39;etichettatura lotto di virus. </li><li> Ricostituire il liofilizzato Alexa Fluor 594 (AF594) esteri succinimidile a 1 mm di etichettatura tampone immediatamente prima della reazione di marcatura. Fluorocromi altro dal Alexa Fluor colorante serie può essere utilizzato in base alle proprie esigenze. Ridurre al minimo l&#39;esposizione alla luce a partire da questo passo. </li><li> Aggiungere 100μl del colorante AF594 1mm a virus diluito mescolando delicatamente con la punta della pipetta. </li><li> Incubare la miscela di reazione etichettatura a temperatura ambiente per 1 ora al buio. Miscelare inversioni dolce ogni 15 minuti. </li><li> Spin brevemente il tubo in una centrifuga da tavolo e aggiungere 100μl di reagente stop alla miscela di reazione mescolando delicatamente con la punta della pipetta. </li><li> Incubare a temperatura ambiente per un&#39;altra ora al buio. Miscelare inversioni dolce ogni 15 minuti. </li></ol> 2. Purificante Alexa Fluor etichettati virus della dengue <ol><li> Nel frattempo, equilibrare la colonna di purificazione con tampone di scelta. In questo esperimento, un PD-10 colonna è equilibrata con 25 ml di tampone HNE (Hepes 5 mm, 150 mm di NaCl, 0,1 mm EDTA), pH 7,4, prima dell&#39;uso. </li><li> Applicare il virus etichettati per la parte superiore della colonna e iniziare a raccogliere le flow-through, una volta che il virus entra nella matrice etichettato. Riempire la colonna con tampone HNE una volta che tutti i virus etichettato è entrato nella matrice. Scartare il primo di 2,5 ml flow-through e raccogliere i prossimi 2 ml di virus frazione etichettati. </li><li> Aliquota e memorizzare purificata AF594 virus della dengue etichettati a -80 ° C, lontano da fonti di luce. </li></ol><ol><li> Scongelare uno aliquota e determinare il titolo del virus etichettato con il saggio della placca prima di utilizzare il lotto di virus etichettati. </li><li> Semi di 5x10 4 per bene di cellule Vero, coltivate in M-199 terreno di coltura, in un 4-pozzetti con un coprioggetto sul fondo di ben un giorno prima dell&#39;infezione. </li><li> Rimuovere il surnatante cultura in bene e infettare le cellule con molteplicità di infezione di 1 del virus etichettati in volume 100μl (diluito in M-199 terreno di mantenimento, come richiesto) per 10 minuti a 37 ° C. </li><li> Rimuovere il inoculums e lavare i coprioggetti due volte in 1xPBS. </li><li> Fissare le cellule nel 3% paraformalydehyde per 30 minuti. </li><li> Lavare i coprioggetti 3 volte in 1xPBS. </li><li> Permeabilize le cellule con una soluzione di permeabilizzazione contenente saponina 0,1% e il 5% BSA in 1xPBS per 30 minuti. </li><li> Incubare le cellule con diluito centrifugazione-sopranatante chiarificato di 3H5 cultura anticorpi monoclonali ibridomi per 1 ora in camera umida, al riparo dalla luce. </li><li> Lavare i coprioggetti 3 volte in tampone di lavaggio (1xPBS contenente cloruro di calcio 1mm, cloruro di magnesio 1 mM e 0,1 saponina%). </li><li> Incubare le cellule con AF488 anti-topo IgG, 1:100 diluito in soluzione permeabilizzazione, per 45 minuti in camera umida, al riparo dalla luce. </li><li> Lavare i coprioggetti 3 volte in tampone di lavaggio e risciacquo, una volta in acqua deionizzata. </li><li> Tamponare il bordo del coprioggetti contro un tovagliolo di carta per drenare l&#39;acqua in eccesso e montare su vetrino con 8μl Mowiol 4-88 DABCO contenente 2,5%. </li><li> Lasciare che la soluzione di montaggio per impostare notte a 4 ° C prima di visualizzare utilizzando un microscopio confocale Zeiss. Acquisizioni sequenziale deve essere eseguito eccitante fluoroforo alla volta e il passaggio tra i rivelatori in concomitanza. </li><li> Le immagini sono poi analizzate per la co-localizzazione delle proteine ​​e degli anticorpi colorazione con il virus etichettato con il software LSM Zen Zeiss per stimare il grado di etichettatura. </li></ol> 3. Rappresentante Risultati Un esempio del rendimento del virus dengue marcato con colorante AF594 è illustrato nella figura 2. Normalmente, a meno di 10 volte cadere dal titolo iniziale dovrebbe essere osservate le seguenti etichette di successo. Tuttavia, va notato che tutti i tamponi devono essere preparati freschi per l&#39;etichettatura per avere successo e Alexa Fluor esteri succinimidile deve essere utilizzato immediatamente dopo la ricostituzione come idrolizzano in acidi liberi reattivi in soluzioni acquose 8. Successivamente, il virus etichettato deve essere controllato per fluorescenza sufficiente prima dell&#39;uso negli esperimenti. Un semplice test di immunofluorescenza è stato fatto su cellule Vero e il grado di etichettatura può essere definita sulla base dei co-localizzazione del virus etichettati con anti-E colorazione delle proteine ​​anticorpali. SeverAl cellule sono state esaminate e una tipica immagine confocale è mostrata in Figura 3. Co-localizzazione di analisi delle immagini utilizzando il software LSM Zen dimostrato sovrapposizione coefficienti che variano dal 0,65-0,8, il che suggerisce che circa il 65 l&#39;80% dei virioni sono stati etichettati con il colorante. <img src="/files/ftp_upload/3168/3168fig1.jpg" alt="Figura 1" /> Figura schema 1.Overall raffigurante la Alexa Fluor etichettatura tintura di procedura virus della dengue. In primo luogo, i buffer pertinenti e virus dengue purificato sono preparati. Il Alexa Fluor colorante è ricostituito e ha aggiunto al virus dengue diluito in tampone di etichettatura. La reazione è allora fermato dopo 1 ora con l&#39;aggiunta di reagente di arresto. Successivamente, il virus marcato è purificata attraverso una colonna di esclusione dimensioni per rimuovere colorante libero. Infine, il virus etichettato è ri-titolato mediante test placca e testati per fluorescenza. <img src="/files/ftp_upload/3168/3168fig2.jpg" alt="Figura 2" /> Figura 2. Il numero medio di virioni vitali (pfu / ml) su un determinato dosaggio placca prima e dopo l&#39;AF594 etichettatura. Un&#39;aliquota del virus AF594 etichettato dengue è scongelato e ri-titolato con il saggio della placca e si vede in genere meno di 10 volte caduta da il titolo di partenza. Le barre di errore indicano la deviazione standard di duplicati. <img src="/files/ftp_upload/3168/3168fig3.jpg" alt="Figura 3" /> Figura 3. Co-localizzazione di AF594 etichette con le proteine ​​del virus dengue E su cellule Vero. Cellule coltivate su vetrini del giorno precedente sono stati infettati con AF594 dengue etichettate MOI di 1 per 10 minuti a 37 ° C. Le cellule sono state successivamente fissa e marcato con anticorpi anti-E, ed esaminati per colocalizzazione della proteina E (verde) e AF594 etichettatura (rosso). Segnali fluorescenti sono state visualizzate sotto ingrandimento 63x con Zeiss LSM 710 microscopio confocale. Barra di scala è 10μm. Giallo indicare le aree di colocalizzazione, come mostrato nel riquadro.

<ol>
	<li>Olenych, S. G., Claxton, N. S., Ottenberg, G. K. &. a. m. p. ;. a. m. p., Davidson, M. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Fluorescent+Protein+Color+Palette.">The Fluorescent Protein Color Palette.</a> Current Protocols in Cell Biology. 21.5, 1-34 (2007).</li><li>Panchuk-Voloshina, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Alexa+dyes,+a+series+of+new+fluorescent+dyes+that+yield+exceptionally+bright,+photostable+conjugates.">Alexa dyes, a series of new fluorescent dyes that yield exceptionally bright, photostable conjugates.</a> J Histochem Cytochem. 47, 1179-1188 (1999).</li><li>Shaner, N. C., Patterson, G. H., Davidson, M. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Advances+in+fluorescent+protein+technology.">Advances in fluorescent protein technology.</a> J Cell Sci. 120, 4247-4260 (2007).</li><li>Lippincott-Schwartz, J., Patterson, G. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+and+use+of+fluorescent+protein+markers+in+living+cells.">Development and use of fluorescent protein markers in living cells.</a> Science. 300, 87-91 (2003).</li><li>Kuhn, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structure+of+dengue+virus:+implications+for+flavivirus+organization,+maturation,+and+fusion.">Structure of dengue virus: implications for flavivirus organization, maturation, and fusion.</a> Cell. 108, 717-725 (2002).</li><li>Aguilar, J. S., Roy, D., Ghazal, P., Wagner, E. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dimethyl+sulfoxide+blocks+herpes+simplex+virus-1+productive+infection+in+vitro+acting+at+different+stages+with+positive+cooperativity.+Application+of+micro-array+analysis.">Dimethyl sulfoxide blocks herpes simplex virus-1 productive infection in vitro acting at different stages with positive cooperativity. Application of micro-array analysis.</a> BMC Infect Dis. 2, 9-9 (2002).</li><li>Zhang, S. L., Tan, H. C., Hanson, B. J., Ooi, E. E. <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+method+for+Alexa+Fluor+dye+labelling+of+dengue+virus.">A simple method for Alexa Fluor dye labelling of dengue virus.</a> J Virol Methods. 167, 172-177 (2010).</li><li>. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Alexa Fluor Succinimidyl Esters (Molecular Probes. , (2009).</li><li>Huang, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analysis+of+proteins+stained+by+Alexa+dyes.">Analysis of proteins stained by Alexa dyes.</a> Electrophoresis. 25, 779-784 (2004).</li><li>Freistadt, M. S., Eberle, K. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fluorescent+poliovirus+for+flow+cytometric+cell+surface+binding+studies.">Fluorescent poliovirus for flow cytometric cell surface binding studies.</a> J Virol Methods. 134, 1-7 (2006).</li></ol>

Anche se AF594 colorante è stato utilizzato in questo rapporto, una vasta gamma di fluorofori nella serie Alexa Fluor succinimidile esteri è disponibile con la chimica etichettatura simili. Questo potrebbe estendere l&#39;applicazione dell&#39;etichettatura di là di imaging. Citometria a flusso può essere usato come alternativa alla microscopia confocale per valutare il grado di etichettatura per fluorofori che può essere eccitato e individuate dalla macchina FACS. Alexa Fluor coloranti...

<table border="1"><tbody><tr><th> Nome del reagente </th><th> Azienda </th><th> Numero di catalogo </th><th> Commenti (opzionale) </th></tr><tr><td> Bicarbonato di sodio </td><td> Sigma-Aldrich </td><td> S6297 </td><td></td></tr><tr><td> Idrossilammina </td><td> Sigma-Aldrich </td><td> 159417 </td><td></td></tr><tr><td> Idrossido di sodio </td><td> Merck </td><td> 106498 </td><td></td></tr><tr><td> AF594 esteri succinimidile </td><td> Sonde molecolari, Invitrogen </td><td> A20004 </td><td></td></tr><tr><td> PD-10 colonna </td><td> GE Healthcare </td><td> 17-0851-01 </td><td></td></tr><tr><td> Hepes </td><td> Sigma-Aldrich </td><td> H6147 </td><td></td></tr><tr><td> NaCl </td><td> Sigma-Aldrich </td><td> S3014 </td><td></td></tr><tr><td> EDTA </td><td> Sigma-Aldrich </td><td> E9884 </td><td></td></tr><tr><td> M-199 </td><td> Invitrogen </td><td> 11150 </td><td></td></tr><tr><td> FBS </td><td> Hyclone </td><td> SH30070.03 </td><td></td></tr><tr><td> 4 pozzetti </td><td> Nunc </td><td> 176740 </td><td></td></tr><tr><td> Coprioggetto </td><td> Einst </td><td> 0111520 </td><td></td></tr><tr><td> Vetrino da microscopio </td><td> Vela Marca </td><td> 7105 </td><td></td></tr><tr><td> Ibridomi 3H5 </td><td> ATCC </td><td> HB46 </td><td></td></tr><tr><td> 10x PBS </td><td> 1 Base </td><td> BUF-2040-10X1L </td><td></td></tr><tr><td> Saponina </td><td> Sigma-Aldrich </td><td> S4521 </td><td></td></tr><tr><td> BSA </td><td> Sigma-Aldrich </td><td> A7906 </td><td></td></tr><tr><td> Cloruro di magnesio </td><td> Sigma-Aldrich </td><td> M2670 </td><td></td></tr><tr><td> Cloruro di calcio </td><td> Sigma-Aldrich </td><td> C3306 </td><td></td></tr><tr><td> Paraformaldehye </td><td> Sigma-Aldrich </td><td> 15,812-7 </td><td></td></tr><tr><td> Mowiol 4-88 </td><td> Calbiochem </td><td> 475904 </td><td></td></tr><tr><td> DABCO </td><td> Sigma-Aldrich </td><td> D27802 </td><td></td></tr><tr><td> Centrifuga da tavolo </td><td> Eppendorf </td><td> 5424 </td><td></td></tr><tr><td> Microscopio confocale </td><td> Zeiss </td><td> LSM 710 </td><td></td></tr></tbody></table> Per preparare M-199 terreno di coltura, aggiungere 50 ml di FBS, 5 ml di piruvato di sodio e 5 ml di aminoacidi non essenziali a 500 ml di M-199, filtro sterile. Per preparare M-199 terreno di mantenimento, aggiungere 15 ml di FBS, 5 ml di piruvato di sodio e 5 ml di aminoacidi non essenziali a 500 ml di M-199, filtro sterile.

visualizing dengue virus through alexa fluor labeling

Gli eventi presto l&#39;interazione tra virus e cellule possono avere un&#39;influenza profonda sul risultato di infezione. Determinare i fattori che influenzano questa interazione potrebbe portare a una migliore comprensione della patogenesi della malattia e quindi influenzare vaccino terapeutico o di progettazione. Quindi, lo sviluppo di metodi per sondare questa interazione potrebbe essere utile. Più recenti progressi della fluorofori sviluppo 1-3 e tecnologia di imaging 4 può essere sfruttato per migliorare le nostre attuali conoscenze sulla patogenesi dengue e quindi aprire la strada per ridurre i milioni di dengue che si verificano ogni anno. Il virus dengue ha un involucro esterno costituito da un&#39;impalcatura di 90 busta glicoproteina (E) dimeri che proteggono il serbatoio nucleocapside, che contiene un singolo filamento positivo di RNA del genoma 5. Le subunità proteiche identiche sulla superficie del virus può quindi essere etichettato con un colorante reattivo ammina e visualizzati tramite microscopia a immunofluorescenza. Qui, presentiamo un semplice metodo di etichettatura dei virus della dengue con Alexa Fluor succinimidile estere colorante sciolto direttamente in un buffer di bicarbonato di sodio che ha dato virus altamente vitali dopo etichettatura. Non esiste una procedura standardizzata per l&#39;etichettatura dei virus vivo e protocollo del produttore esistente per l&#39;etichettatura delle proteine ​​di solito richiede la ricostituzione di colorante in dimetilsolfossido. La presenza di dimetilsolfossido, anche in piccole quantità, in grado di bloccare l&#39;infezione produttiva da virus e anche indurre citotossicità delle cellule 6. L&#39;esclusione dell&#39;uso di dimetilsolfossido in questo protocollo così ridotto questa possibilità. Alexa Fluor coloranti hanno fotostabilità superiore ed è meno sensibile al pH rispetto ai coloranti comuni, come la fluoresceina e rodamina 2, che li rende ideali per gli studi sulla captazione cellulare e il trasporto endosomali del virus. La coniugazione di Alexa Fluor colorante non ha influenzato il riconoscimento dei virus della dengue etichettati da virus-specifici anticorpi ed i suoi recettori putativi in cellule ospiti 7. Questo metodo potrebbe avere applicazioni utili in studi virologici.

Watch this Scientific Journal Video about Visualizing Dengue Virus through Alexa Fluor Labeling at JoVE.com

Visualizing Dengue Virus through Alexa Fluor Labeling

Approfittando dei progressi nello sviluppo fluoroforo e tecnologia di imaging, un metodo semplice di Alexa Fluor etichettatura dei virus dengue è stato ideato per visualizzare le prime interazioni tra virus e cellule.

Visualizzazione virus Dengue attraverso Alexa Fluor Etichettatura

The early events in the interaction between virus and cell can have profound influence on the outcome of infection. Determining the factors 
that ...

visualizing-dengue-virus-through-alexa-fluor-labeling

Research

JoVE Journal

Immunology and Infection

13.8K Views.  DSO National Laboratories. Approfittando dei progressi nello sviluppo fluoroforo e tecnologia di imaging, un metodo semplice di Alexa Fluor etichettatura dei virus dengue è stato ideato per visualizzare le prime interazioni tra virus e cellule.

Video: Visualizing Dengue Virus through Alexa Fluor Labeling

Alphavirus Transducing System:  Tools for Visualizing Infection in Mosquito Vectors

Alphavirus transducing systems (ATSs) are important tools for expressing genes of interest (GOI) during infection. ATSs are derived from cDNA clones of mosquito-borne RNA viruses (genus Alphavirus; family Togaviridae). The Alphavirus genus contains about 30 different mosquito-borne virus species. Alphaviruses are enveloped viruses and contain single-stranded RNA genomes (~11.7 Kb). Alphaviruses transcribe a subgenomic mRNA that encodes the structural proteins of the virus required for encapsidation of the genome and maturation of the virus. Alphaviruses are usually highly lytic in vertebrate cells, but persistently infect susceptible mosquito cells with minimal cytopathology. These attributes make them excellent tools for gene expression in mosquito vectors. The most common ATSs in use are derived from Sindbis virus (SINV). The broad species tropism of SINV allows for infection of insect, avian, and mammalian cells8. However, ATSs have been derived from other alphaviruses as well9,10,20. Foreign gene expression is made possible by the insertion of an additional viral subgenomic RNA initiation site or promoter. ATSs in which an exogenous gene sequence is positioned 5' to the viral structural genes is used for stable protein expression in insects. ATSs, in which a gene sequence is positioned 3' to the structural genes, is used to trigger RNAi and silence expression of that gene in the insect.
ATSs have proven to be valuable tools for understanding vector-pathogen interactions, molecular details of viral replication and maintenance infectious cycles3,4,11,19,21. In particular, the expression of fluorescent and bioluminescent reporters has been instrumental tracking the viral infection in the vector and virus transmission5,14-16,18. Additionally, the vector immune response has been described using two strains of SINV engineered to express GFP2,9.
Here, we present a method for the production of SINV containing a fluorescent reporter (GFP) from the cDNA infectious clone. Infectious, full-length RNA is transcribed from the linearized cDNA clone. Infectious RNA is introduced into permissive target cells by electroporation. Transfected cells generate infectious virus particles expressing the GOI. Harvested virus is used to infect mosquitoes, as described here, or other host species (not shown herein). Vector competence is assessed by detecting fluorescence outside the midgut or by monitoring virus transmission7. Use of a fluorescent reporter as the GOI allows for convenient estimation of virus spread throughout a cell culture, for determination of rate of infection, dissemination in exposed mosquitoes, virus transmission from the mosquito and provides a rapid gauge of vector competence.

Methods for using alphavirus transducing systems to express fluorescent reporters in vitro and in adult mosquitoes are described. This technique may be adapted to express any protein of interest in lieu of or in addition to a reporter.

Sistema alfavirus trasduzione: Strumenti per la visualizzazione di infezione in Vettori Mosquito

Alphavirus transducing systems (ATSs) are important tools for expressing genes of interest (GOI) during infection.  ATSs are derived from cDNA clones of ...

Ricerca

Immunologia e infezioni

A Method for Labeling Vasculature in Embryonic Mice

The establishment of a functional blood vessel network is an essential part of organogenesis, and is required for optimal organ function. For example, in the thymus proper vasculature formation and patterning is essential for thymocyte entry into the organ and mature T-cell exit to the periphery. The spatial arrangement of blood vessels in the thymus is dependent upon signals from the local microenvironment, namely thymic epithelial cells (TEC). Several recent reports suggest that disruption of these signals results in thymus blood vessel defects 1,2. Previous studies have described techniques used to label the neonatal and adult thymus vasculature 1,2. We demonstrate here a technique for labeling blood vessels in the embryonic thymus. This method combines the use of FITC-dextran or Griffonia (Bandeiraea) Simplicifolia Lectin I (GSL 1 - isolectin B4) facial vein injections and CD31 antibody staining to identify thymus vascular structures and PDGFR-&beta; to label thymic perivascular mesenchyme 3-5. The option of using cryosections or vibratome sections is also provided. This protocol can be used to identify thymus vascular defects, which is critical for defining the roles of TEC-derived molecules in thymus blood vessel formation. As the method labels the entire vasculature, it can also be used to analyze the vascular networks in multiple organs and tissues throughout the embryo including skin and heart 6-10.

This article describes a method for labeling embryonic skin and thymus blood vessels.

Un metodo per l&#39;etichettatura vasi embrionali nei topi

The establishment of a functional blood vessel network is an essential part of organogenesis, and is required for optimal organ function. For example, in ...

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 ...

Evaluation of the Efficacy And Toxicity of RNAs Targeting HIV-1 Production for Use in Gene or Drug Therapy

Small RNA therapies targeting post-integration steps in the HIV-1 replication cycle are among the top candidates for gene therapy and have the potential to be used as drug therapies for HIV-1 infection. Post-integration inhibitors include ribozymes, short hairpin (sh) RNAs, small interfering (si) RNAs, U1 interference (U1i) RNAs and RNA aptamers. Many of these have been identified using transient co-transfection assays with an HIV-1 expression plasmid and some have advanced to clinical trials. In addition to measures of efficacy, small RNAs have been evaluated for their potential to affect the expression of human RNAs, alter cell growth and/or differentiation, and elicit innate immune responses. In the protocols described here, a set of transient transfection assays designed to evaluate the efficacy and toxicity of RNA molecules targeting post-integration steps in the HIV-1 replication cycle are described. We have used these assays to identify new ribozymes and optimize the format of shRNAs and siRNAs targeting HIV-1 RNA. The methods provide a quick set of assays that are useful for screening new anti-HIV-1 RNAs and could be adapted to screen other post-integration inhibitors of HIV-1 replication.

Methods to evaluate the efficacy and toxicity of RNA molecules targeting post-integration steps of the HIV-1 replication cycle are described. These methods are useful for screening new molecules and optimizing the format of existing ones.

Valutazione della efficacia e la tossicità degli RNA targeting HIV-1 Produzione per l&#39;uso in gene terapia farmacologica

Small RNA therapies targeting post-integration steps in the HIV-1 replication cycle are among the top candidates for gene therapy and have the potential ...

Rescue and Characterization of Recombinant Virus from a New World Zika Virus Infectious Clone

Infectious cDNA clones allow for genetic manipulation of a virus, thus facilitating work on vaccines, pathogenesis, replication, transmission and viral evolution. Here we describe the construction of an infectious clone for Zika virus (ZIKV), which is currently causing an explosive outbreak in the Americas. To prevent toxicity to bacteria that is commonly observed with flavivirus-derived plasmids, we generated a two-plasmid system which separates the genome at the NS1 gene and is more stable than full-length constructs that could not be successfully recovered without mutations. After digestion and ligation to join the two fragments, full-length viral RNA can be generated by in vitro transcription with T7 RNA polymerase. Following electroporation of transcribed RNA into cells, virus was recovered that exhibited similar in vitro growth kinetics and in vivo virulence and infection phenotypes in mice and mosquitoes, respectively.

This protocol describes the recovery of infectious Zika virus from a two-plasmid infectious cDNA clone.

Salvataggio e caratterizzazione del virus ricombinante da un nuovo mondo Clone infettivo del virus Zika

Infectious cDNA clones allow for genetic manipulation of a virus, thus facilitating work on vaccines, pathogenesis, replication, transmission and viral ...

Visualizing Macrophage Extracellular Traps Using Confocal Microscopy

A primary method used to define the presence of neutrophil extracellular traps (NETs) is confocal microscopy. We have modified established confocal microscopy methods to visualize macrophage extracellular traps (METs). These extracellular traps are defined by the presence of extracellular chromatin with co-expression of other components such as granule proteases, citrullinated histones, and peptidyl arginase deiminase (PAD). The expression of METs is generally measured after exposure to a stimulus and compared to un-stimulated samples. Samples are also included for background and isotype control. Cells are analyzed using well-defined image analysis software. Confocal microscopy may be used to define the presence of METs both in vitro and in vivo in lung tissue.

Macrophage extracellular traps are a newly described entity. This article will concentrate on confocal microscopy methods and how they are visualized in vitro and in vivo from lung samples.

Visualizzazione del macrofago trappole extracellulari mediante microscopia confocale

A primary method used to define the presence of neutrophil extracellular traps (NETs) is confocal microscopy. We have modified established confocal ...

Visualizing Intracellular SNARE Trafficking by Fluorescence Lifetime Imaging Microscopy

Soluble N-ethylmaleimide sensitive fusion protein (NSF) attachment protein receptor (SNARE) proteins are key for membrane trafficking, as they catalyze membrane fusion within eukaryotic cells. The SNARE protein family consists of about 36 different members. Specific intracellular transport routes are catalyzed by specific sets of 3 or 4 SNARE proteins that thereby contribute to the specificity and fidelity of membrane trafficking. However, studying the precise function of SNARE proteins is technically challenging, because SNAREs are highly abundant and functionally redundant, with most SNAREs having multiple and overlapping functions. In this protocol, a new method for the visualization of SNARE complex formation in live cells is described. This method is based on expressing SNARE proteins C-terminally fused to fluorescent proteins and measuring their interaction by F&#246;rster resonance energy transfer (FRET) employing fluorescence lifetime imaging microscopy (FLIM). By fitting the fluorescence lifetime histograms with a multicomponent decay model, FRET-FLIM allows (semi-)quantitative estimation of the fraction of the SNARE complex formation at different vesicles. This protocol has been successfully applied to visualize SNARE complex formation at the plasma membrane and at endosomal compartments in mammalian cell lines and primary immune cells, and can be readily extended to study SNARE functions at other organelles in animal, plant, and fungal cells.

This protocol describes a new method allowing for the quantitative visualization of complex formation of SNARE proteins, based on F&#246;rster resonance energy transfer, and fluorescence lifetime imaging microscopy.

Visualizing intracellulare rullante traffico da fluorescenza Lifetime Imaging microscopia

Soluble N-ethylmaleimide sensitive fusion protein (NSF) attachment protein receptor (SNARE) proteins are key for membrane trafficking, as they catalyze ...

A Simple Flow Cytometry Based Assay to Determine In Vitro Antibody Dependent Enhancement of Dengue Virus Using Zika Virus Convalescent Serum

Antibody dependent enhancement of infection has been shown to play a major role in Dengue viral pathogenesis. Traditional assays that measure the capacity of antibodies or serum to enhance infection in impermissible cell lines have relied on using viral output in the media followed by plaque assays to quantify infection. More recently, these assays have examined Dengue virus (DENV) infection in the cell lines using fluorescently labeled antibodies. Both these approaches have limitations that restrict the widespread use of these techniques. Here, we describe a simple in vitro assay using Dengue virus reporter viral particles (RVPs) that express green fluorescent protein and K562 cells to examine antibody dependent enhancement (ADE) of DENV infection using serum that was obtained from rhesus macaques 16 weeks after infection with Zika virus (ZIKV). This technique is reliable, involves minimal manipulation of cells, does not involve the use of live replication competent virus, and can be performed in a high throughput format to get a quantitative readout using flow cytometry. Additionally, this assay can be easily adapted to examine antibody dependent enhancement (ADE) of other flavivirus infections such as Yellow Fever virus (YFV), Japanese Equine Encephalitis virus (JEEV), West Nile virus (WNV) etc. where RVPs are available. The ease of setting up the assay, analyzing the data, and interpreting results makes it highly amenable to most laboratory settings.

A Simple Flow Cytometry Based Assay to Determine In Vitro Antibody Dependent Enhancement of Dengue Virus Using Zika Virus Convalescent Serum

We describe a simple and easy protocol for measuring antibody dependent enhancement of infection by Zika virus convalescent serum using Dengue virus Reporter Viral Particles.

Base di un semplice flusso Cytometry Assay per determinare In Vitro dell'anticorpo dipendente valorizzazione del Virus di febbre rompiossa utilizzando siero convalescente Zika Virus

Antibody dependent enhancement of infection has been shown to play a major role in Dengue viral pathogenesis. Traditional assays that measure the capacity ...

Rapid, Safe, and Simple Manual Bedside Nucleic Acid Extraction for the Detection of Virus in Whole Blood Samples

The rapid diagnosis of an infection is essential for the outbreak management, risk containment, and patient care. We have previously shown a method for the rapid bedside inactivation of the Ebola virus during blood sampling for safe nucleic acid (NA) tests by adding a commercial lysis/binding buffer directly into the vacuum blood collection tubes. Using this bedside inactivation approach, we have developed a safe, rapid, and simplified bedside NA extraction method for the subsequent detection of a virus in lysis/binding buffer-inactivated whole blood. The NA extraction is directly performed in the blood collection tubes and requires no equipment or electricity. 
After the blood is collected into the lysis/binding buffer, the contents are mixed by flipping the tube by hand, and the mixture is incubated for 20 min at room temperature. Magnetic glass particles (MGPs) are added to the tube, and the contents are mixed by flipping the collection tube by hand. The MGPs are then collected on the side of the blood collection tube using a magnetic holder or a magnet and a rubber band. The MGPs are washed three times, and after the addition of elution buffer directly into the collection tube, the NAs are ready for NA tests, such as qPCR or isothermal loop amplification (LAMP), without the removal of the MGPs from the reaction. The NA extraction method is not dependent on any laboratory facilities and can easily be used anywhere (e.g., in field hospitals and hospital isolation wards). When this NA extraction method is combined with LAMP and a portable instrument, a diagnosis can be obtained within 40 min of the blood collection.

Here, we present a protocol for the rapid virus nucleic acid extraction from the virus-inactivated whole blood. The extraction is performed directly in the blood collection tubes and requires no equipment or electricity. The method is not dependent on laboratory facilities and can be used anywhere (e.g., in field hospitals).

Rapido, sicuro e semplice manuale sul comodino estrazione dell'acido nucleico per la rilevazione del Virus in campioni di sangue intero

The rapid diagnosis of an infection is essential for the outbreak management, risk containment, and patient care. We have previously shown a method for ...

Measuring Dengue Virus RNA in the Culture Supernatant of Infected Cells by Real-time Quantitative Polymerase Chain Reaction

At present, real-time polymerase chain reaction (PCR) technology is an indispensable tool for the detection and quantification of viral genomes in research laboratories, as well as for molecular diagnosis, because of its sensitivity, specificity, and convenience. However, in most cases, the quantitative PCR (qPCR) assay generally used to detect virus infection has relied on the purification of viral nucleic acid prior to the PCR step. In this study, the fluorescence-based reverse transcription qPCR (RT-qPCR) assay is developed through the combination of a processing buffer and a one-step RT-PCR reagent so that the whole process, from the harvest of the culture supernatant of virus-infected cells until real-time detection, can be performed without viral RNA purification. The established protocol enables the quantification of a wide range of RNA concentrations of dengue virus (DENV) within 90 min. In addition, the adaptability of the direct RT-qPCR assay to the evaluation of an antiviral agent is demonstrated by an in vitro experiment using a previously reported DENV inhibitor, mycophenolic acid (MPA). Moreover, other RNA viruses, including yellow fever virus (YFV), Chikungunya virus (CHIKV), and measles virus (MeV), can be quantified by direct RT-qPCR with the same protocol. Therefore, the direct RT-qPCR assay described in this report is useful for monitoring RNA virus replication in a simple and rapid manner, which will be further developed into a promising platform for a high-throughput screening study and clinical diagnosis.

Real-time quantitative polymerase chain reaction analysis combined with reverse transcription (RT-qPCR) has been widely used to measure the level of RNA virus infections. Here we present a direct RT-qPCR assay, which does not require an RNA purification step, developed for the quantification of several RNA viruses, including dengue virus.

RNA del Virus di febbre rompiossa nel supernatante di coltura delle cellule infettate da reazione a catena della polimerasi quantitativa in tempo reale di misurazione

Visualizzazione virus Dengue attraverso Alexa Fluor Etichettatura

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