This work was funded by both the&#160;Canadian Institutes of Health Research&#160;and&#160;Natural Sciences and the Engineering Research Council of Canada. M. Bisaillon is a Chercheur Boursier Senior from the Fonds de Recherche en Sant&#233; du Qu&#233;bec and also a member of the Centre de Recherche Clinique du Centre Hospitalier Universitaire de Sherbrooke. We thank Dr. Ralf Bartenschlager for the generous gift of the HCV replicon system. We also thank Dr. Charles Rice and Dr. Daniel Lamarre for kindly providing the hepatic cell line. We also want to thank Guillaume Tremblay for technical assistance.

1. Le celle di preparazione e trattamento <ol><li> In una piastra a 24 pozzetti, mettere una copertura in vetro sul fondo dei pozzetti. Lavare ogni pozzetto con tampone fosfato (PBS). </li><li> Seme Huh-7 cellule di supporto HCV replicone subgenomico in ciascun pozzetto ad una densità di 5 × 10 4 cellule / pozzetto. Se non esiste alcun trattamento per fare, le cellule possono essere seminate ad una densità maggiore. Il terreno di coltura è Dulbecco Modified Eagle Medium (DMEM) supplementato con 10% siero fetale bovino (FBS), 2 mM L-glutammina, 1 mM sodio piruvato e 250 mg / ml G-418 (per mantenere la replicone). </li><li> Incubare a 37 ° C e 5% CO 2. Il giorno dopo, trattare le cellule con 3 mM di HLF purificata da latte umano. </li></ol> 2. Paraformaldeide / saccarosio Preparazione (12% PAF e il 12% di saccarosio) <ol><li> Mettere 500 ml di PBS in un becher e riscaldarla tra 20 ° C e 30 ° C. Sciogliere 60 g di saccarosio in PBS. Sciogliere 60 g di paraformaldeide (PAF) nel / soluzione di saccarosio PBS. Aggiungere lentamente NaOH 2N (3 a 7 ml) fino a soluzione limpida ottenuta. </li><li> Regolare il pH a 7,4 con HCl. Volume completa a 500 ml con PBS. Filtra per gravità su filtro Whatman. Prima dell&#39;uso, diluire con PBS ad una concentrazione finale di 4% e il 4% PAF saccarosio (memoria 4 ° C per 1 settimana massimo). </li></ol> 3. immunofluorescenza <ol><li> Al tempo desiderato (0 ore, 2 ore o 24 ore di trattamento), lavare le cellule due volte (1 minuto) con PBS e fissare con PBS / 4% PAF / 4% di saccarosio per 20 minuti a temperatura ambiente. Le cellule sono tipicamente a 30-40% di confluenza e sono utilizzati 1,5x10 5 cellule. </li><li> Permeabilize le cellule con 0,15% triton X-100 in PBS per 5 minuti a RT e blocco in siero normale di capra 10% (NGS) in PBS per 20 min. </li><li> Diluire l&#39;anticorpo primario della proteina di interesse in 10% NGS (esempio: principale del mouse anticorpo anti-HLF diluito 1: 1000). Applicare l&#39;anticorpo primario alle cellule. </li><li> Dopo 4 ore a temperatura ambiente o O /N a 4 ° C, lavare le cellule tre volte per 5 min a 10% NGS. Macchiare le cellule con anticorpo secondario marcato con fluorocromo diluito in 10% NGS (esempio: Fluorocromo con lunghezza d&#39;onda di eccitazione di 488 nm legata ad un anticorpo anti-topo 1: 1.000) per 1 ora a RT al buio. </li><li> Lavare le cellule una volta per 5 minuti con PBS e macchiare le cellule con 4 &#39;, 6-diamidino-2-fenilindolo (DAPI) (1 mg / ml) per 15 minuti a RT al buio. </li><li> Lavare le cellule due volte per 5 minuti con PBS. Monte coprire occhiali sul mezzo di montaggio SlowFade prima visualizzazione tramite microscopia. Le cellule sono ora pronti per l&#39;analisi da epifluorescenza o microscopia confocale. </li><li> Utilizzare diversi controlli per assicurare la specificità del rilevamento. Ad esempio, tutti gli anticorpi possono essere omessi per rilevare autofluorescenza. L&#39;anticorpo primario può essere omesso per determinare la colorazione non specifica da parte dell&#39;anticorpo secondario. Infine, se possibile, controllare cellule o tessuti che non lo fannoesprimono la molecola di interesse può anche essere usato. </li><li> Visualizzare i campioni utilizzando appropriati microscopio a fluorescenza (epifluorescenza o confocale) e set di filtri appropriati per l&#39;etichetta fluorescente usato. Le diapositive possono anche essere memorizzati in una scatola di diapositive a &lt;-20 ° C per un esame successivo. </li></ol> 4. microscopia a fluorescenza <ol><li> Conservare i campioni al buio per evitare photobleaching. Accendere la sorgente di luce del microscopio scelta (epifluorescenza o confocale). </li><li> Accendere il microscopio. Accendere la fotocamera per il microscopio. Mettere il vetrino sul microscopio per la visualizzazione. Aggiungere olio per immersione sul vetrino per aumentare l&#39;apertura numerica della lente obiettivo. </li><li> Selezionare i filtri appropriati. Regolare la messa a fuoco e l&#39;obiettivo appropriato. Regolare le imposte, al fine di rilevare il fluorocromo appropriata ma evitare photobleaching. Mantenere luci ambientali per minimizzare sfondo chiaro quando guardando campione. </li><li> Cambia °filtri e di visualizzare diversi fluorocromi (ad esempio DAPI e per una lunghezza d&#39;onda di eccitazione di 488 nm). Scatta foto con la fotocamera. </li></ol>

<ol>
	<li>Odell, I. D., Cook, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immunofluorescence+techniques.">Immunofluorescence techniques.</a> J Invest Derm. 133 (1), e4 (2013).</li><li>Portugal, J., Waring, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Assignment+of+DNA+binding+sites+for+4&#8217;,6-diamidine-2-phenylindole+and+bisbenzimide+(Hoechst+33258).+A+comparative+footprinting+study.">Assignment of DNA binding sites for 4&#8217;,6-diamidine-2-phenylindole and bisbenzimide (Hoechst 33258). A comparative footprinting study.</a> Biochim Biophys Acta. 949 (2), 158-168 (1988).</li><li>Lichtman, J. W., Conchello, J. -. A. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fluorescence+microscopy.">Fluorescence microscopy.</a> Nat Methods. 2 (12), 910-919 (2005).</li><li>St Croix, C. M., Shand, S. H., Watkins, S. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Confocal+microscopy:+comparisons,+applications,+and+problems.">Confocal microscopy: comparisons, applications, and problems.</a> BioTechniques. 39, S2-S5 (2005).</li><li>Yi, M., Kaneko, S., Yu, D. Y., Murakami, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hepatitis+C+virus+envelope+proteins+bind+lactoferrin.">Hepatitis C virus envelope proteins bind lactoferrin.</a> Journal of virology. 71, 5997-6002 (1997).</li><li>Wakabayashi, H., Oda, H., Yamauchi, K., Abe, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lactoferrin+for+prevention+of+common+viral+infections.">Lactoferrin for prevention of common viral infections.</a> J Inf Chem. 20 (11), 666-671 (2014).</li><li>Picard-Jean, F., Bouchard, S., Lariv&#233;e, G., Bisaillon, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+intracellular+inhibition+of+HCV+replication+represents+a+novel+mechanism+of+action+by+the+innate+immune+Lactoferrin+protein.">The intracellular inhibition of HCV replication represents a novel mechanism of action by the innate immune Lactoferrin protein.</a> Antiviral research. 111, 13-22 (2014).</li><li>Johnson, G. D., Davidson, R. S., McNamee, K. C., Russell, G., Goodwin, D., Holborow, E. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fading+of+immunofluorescence+during+microscopy:+a+study+of+the+phenomenon+and+its+remedy.">Fading of immunofluorescence during microscopy: a study of the phenomenon and its remedy.</a> J Immunol Methods. 55 (2), 231-242 (1982).</li><li>Green Remington, S. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=fluorescent+protein:+a+perspective.">fluorescent protein: a perspective.</a> Protein Sci. 20 (9), 1509-1519 (2011).</li></ol>

The authors declare that they have no competing financial interests.

L&#39;epidemia HCV rimane una minaccia globale, con l&#39;80% dei pazienti contagiati in via di sviluppo una infezione cronica, mettendoli a rischio di cirrosi, insufficienza epatica o epatocarcinoma. Azione diretta agenti antivirali mirati replicazione di HCV e la maturazione rappresentano agenti anti-HCV primi, come recentemente dimostrato dalla approvazione di regolamentazione di due inibitori della proteasi NS3 N-terminale (boceprevir e telaprevir). L&#39;attività anti-HCV HLF è attualmente crede di comportarsi come un inibitore ingresso del virus, rilegatura circolante virioni e impedendo loro di entrare nella loro obiettivo epatocellulare. La nostra ricerca su HLF presenta un romanzo intracellulare meccanismo anti-HCV di azione (distinto da extracellulare virione neutralizzazione) 7. Immunofluorescenza può essere efficacemente utilizzato per visualizzare la distribuzione e / o localizzazione di una molecola bersaglio in un campione biologico. Come si trova in molte altre tecniche di fluorescenza, uno dei pro principaleproblematiche connesse con immunofluorescenza è photobleaching (distruzione fotochimica del fluorocromo) 8. Questa perdita di attività causata da photobleaching può essere aggirata sia impiegando fluorocromi più robusti che sono meno inclini a photobleaching o riducendo l&#39;intensità o lasso di tempo di esposizione alla luce. Inoltre, poiché gli anticorpi non possono attraversare la membrana cellulare, immunofluorescenza è generalmente limitata solo alle cellule fisse. Approcci alternativi per monitorare la distribuzione / localizzazione delle proteine ​​nelle cellule vive quindi generalmente contare sulla espressione di proteine ​​contenenti domini proteici fluorescenti come la proteina fluorescente verde (GFP) 9. Tuttavia, l&#39;aggiunta di tali epitopi GFP è talvolta associata con una modifica nella localizzazione e l&#39;attività delle proteine. Immunofluorescenza può essere utilizzato anche su diversi campioni anche sezioni di tessuto, linee cellulari coltivate, o cellule individuali è anche frequentemente usato per monitorarela localizzazione / distribuzione delle proteine, glicani, e piccole molecole. I principali vantaggi di questa tecnica risiede nella sua semplicità ed il fatto che esso può essere utilizzato in combinazione con altri metodi, non-anticorpo di fluorescenza (ad esempio, colorazione DNA). Inoltre, anticorpi secondari prodotti commercialmente sono relativamente poco costoso e disponibile in una vasta gamma di colori. Nel corso di studio, immunofluorescenza di cellule epatiche che sostengono una HCV replicone subgenomico rivelato una riduzione qualitativa dell&#39;intensità HCV colorazione in seguito al trattamento con HLF. Questa è un&#39;osservazione interessante come questo sistema replicone subgenomico manca HCV E1 ed E2 glicoproteine, che sono i bersagli farmacologici comunemente accettati HLF. A differenza della extracellulare dell&#39;attività virione neutralizzazione, HLF avrebbe bisogno di essere interiorizzato dagli epatociti con infezione da HCV, al fine di mettere in pericolo la replicazione virale. L&#39;utilizzo della microscopia fuorescence ci ha permesso di valumangiato questa possibilità. Al momento di pre-incubazione in epatociti con infezione da HCV, esogeno HLF è stato interiorizzato con successo, e dimostrato di alterare la replicazione virale intracellulare. La comprensione di questo romanzo HLF attività antivirale costituisce un passo in avanti nella comprensione del meccanismo globale (e probabilmente pleiotropico) di inibizione di questa proteina immunità innata.

Immunofluorescence is a technique that uses a fluorescence microscope to visualize the distribution and/or localization of a target molecule in a biological sample. Immunofluorescence relies on the specificity of fluorescent-labelled antibodies against their corresponding antigens within a cell1. It is typically used on tissue sections and cultured cell lines in order to analyze the distribution/localization of various biological molecules such as proteins, nucleic acids and glycans. It should be noted that immunofluorescence is often used in combination with other non-antibody methods of fluorescent staining such as the 4',6-diamidino-2-phenylindole (DAPI) stains which are typically used to label DNA2. Moreover, this technique involves fixation of the cells which allows the analysis of cells at a specific time.
Different types of microscopes can be used to analyze immunofluorescence samples. The simplest is the epifluorescence microscope (Figure 1) for which excitation of the fluorochrome and detection of the fluorescence are done through the same light path3. Because most of the excitation light is transmitted through the sample, only reflected excitatory light can reach the objective together with the emitted light. This approach unfortunately leadsto a frequent high signal to noise ratio.In contrast, confocal microscopy (Figure 2) offers a distinct advantage for increasing optical resolution and contrast by means of adding a spatial pinhole placed at the confocal plane of the lens to eliminate out-of-focus light4. This approach allows the reconstruction of three-dimensional structures from the obtained images. However, since an important fraction of the light from the sample is blocked at the pinhole, long exposures are often required.
There are two classes of immunofluorescence techniques, primary (or direct) and secondary (or indirect). Direct immunofluorescence involves a primary antibody linked with a fluorochrome (Figure 3). This method is less frequently used because it provides lower signal, involves higher cost and less flexibility1. Moreover, such antibodies are generally harder to find commercially. On the other hand, the direct attachment of the fluorochrome to the antibody significantly reduces the number of steps in the procedure, saving time and frequently reducing non-specific background signal. This also limits the possibility of antibody cross-reactivty.
Indirect immunofluorescence involves a primary unlabelled antibody which is specific for the epitope of interest1. A secondary antibody which carries the fluorochrome then recognizes the primary antibody and binds to it (Figure 3). Although indirect immunofluorescence is more complex and time consuming than direct immunofluorescence, it is frequently used because of its high sensitivity and it also provides an amplified signal since more than one secondary antibody can attach to each primary antibody. In addition, a vast array of commercial secondary antibodies is available at affordable prices.
Hepatitis C virus (HCV) is a major public-health problem with 130-170 million individuals chronically infected worldwide. In order to halt the epidemic, therapy against HCV will need to be both effective and widely available. Studies focusing on safe and affordable natural product active against HCV have revealed the antiviral activity of the human Lactoferrin (hLF) protein which binds and neutralizes the circulating virion5. In the current study, investigation of hLF activity on the HCV subgenomic replicon system, which is independent from viral entry and shedding, revealed a distinct antireplicative activity of hLF against HCV. This manuscript presents a study in which immunofluorescence assays were performed to monitor the internalization of hLF, an important component of the immune system6, into hepatic cells. Moreover, we monitored the inhibitory potential of hLF on the intracellular replication of the Hepatitis C virus (HCV).

<table><tbody><tr><td>DMEM</td><td>Wisent</td><td>319-005-CL</td><td></td></tr><tr><td>PAF</td><td>BioShop</td><td>PAR070.1</td><td>Flammable solid, skin irritant, lungs and eyes&amp;nbsp;</td></tr><tr><td>PBS</td><td>Wisent</td><td>311-425-CL</td><td>Without Ca&lt;sup&gt;2+ &lt;/sup&gt;&amp;amp; Mg&lt;sup&gt;2+&lt;/sup&gt;</td></tr><tr><td>NGS</td><td>Wisent</td><td>053-150</td><td></td></tr><tr><td>AlexaFluor 488-labeled anti-mouse</td><td>Invitrogen</td><td>A11017</td><td></td></tr><tr><td>AlexaFluor 568-labeled anti-rabbit</td><td>Invitrogeb</td><td>A21069</td><td></td></tr><tr><td>Wheat germ agglutinin Alexa Fluor 488 conjugate (WGA)</td><td>Invitrogen</td><td>W11261</td><td>Potentially mutagenic</td></tr><tr><td>Anti-NS5A rabbit</td><td>Abcam</td><td>ab2594</td><td></td></tr><tr><td>Anti-hLF mouse</td><td>Abcam</td><td>ab10110</td><td></td></tr><tr><td>SlowFade</td><td>Invitrogen</td><td>S36937</td><td></td></tr><tr><td>Hoechst stain</td><td>Life Techn.</td><td>H1399</td><td>Potentially&amp;nbsp;mutagenic&amp;nbsp;
and carcinogenic</td></tr><tr><td>hLF</td><td>Sigma</td><td>L0520</td><td></td></tr><tr><td>Nikon Eclipse visible/epifluorescence Microscope</td><td>Nikon</td><td>TE2000-E</td><td></td></tr><tr><td>epifluorescence/confocal microscope</td><td>Olympus</td><td>FV1000</td><td></td></tr></tbody></table>

immunofluorescence to monitor the cellular uptake of human lactoferrin and its associated antiviral activity against the hepatitis c virus

Immunofluorescenza è una tecnica di laboratorio comunemente usata per studiare molti aspetti della biologia. Esso è tipicamente usato per visualizzare la distribuzione e / o la localizzazione di una molecola bersaglio in cellule e tessuti. Immunofluorescenza basa sulla specificità degli anticorpi fluorescenti marcati contro loro antigeni corrispondenti all&#39;interno di una cella. Entrambi gli approcci diretti e indiretti immunofluorescenza possono essere utilizzati che si basano sull&#39;uso di anticorpi legati con un fluorocromo. Immunofluorescenza diretta è meno frequentemente utilizzato perché fornisce il segnale più bassa, comporta costi più elevati e meno flessibilità. Al contrario, immunofluorescenza indiretta è più comunemente utilizzato per la sua elevata sensibilità e fornisce un segnale amplificato da più di un anticorpo secondario può collegare a ciascun anticorpo primario. In questo manoscritto, sia epifluorescenza e microscopia confocale sono stati usati per monitorare l&#39;internalizzazione di lattoferrina umana, una componente importante del sistema immunitariosistema, nelle cellule epatiche. Inoltre, abbiamo monitorato il potenziale inibitorio del HLF sulla replica intracellulare del virus dell&#39;epatite C mediante immunofluorescenza. Entrambi i vantaggi e gli svantaggi associati a questi approcci sono discussi.

La capacità del epatica linea cellulare Huh-7 di internalizzare esogena amministrato HLF è stata monitorata mediante immunofluorescenza su una miscroscope confocale. HLF è stato aggiunto al mezzo di coltura e l&#39;internalizzazione stato consentito per 24 ore, dopo di che, extracellulare HLF è stato lavato con PBS e membrana residuo legato HLF stato degradato con un 5 min trattamento tripsina (1 ml). Le cellule sono state reseeded e ha permesso di ri-aderire per 18 ore prima di immunofluorescenza. Al fine di delineare i limiti citoplasmatici, le membrane di cellule sono state colorate con germe di grano agglutinina (WGA) fluorocromi di lunghezza d&#39;onda di eccitazione di 488 nm coniugato. La colorazione HLF è stata ottenuta utilizzando un anticorpo primario anti-HLF e un fluorocromo con una lunghezza d&#39;onda di 633 nm legata all&#39;anticorpo secondario. Al fine di determinare la localizzazione precisa del HLF, cellule sono state ripreso seguente sequenziale 0,5 micron orizzontale sezione trasversale mediante microscopia confocale. La Figura 4 presenta un optical sezione trasversale corrispondente a metà dello spessore cellule. Dopo l&#39;aggiunta di esogeno HLF, una quantità significativa di HLF è stato rilevato all&#39;interno del citoplasma degli epatociti. No HLF è stata osservata all&#39;interno delle cellule non trattate utilizzando le impostazioni strumentali identiche. Insieme, questi dati confermano che epatociti hanno la capacità di acquisire HLF dal loro ambiente extracellulare. La capacità di HLF di inibire la replicazione intracellulare dell&#39;HCV era accanto monitorata con epifluorescenza. Huh-7 cellule di supporto HCV replicone subgenomico (esprimendo HCV NS3, NS4A, NS4B, dell&#39;NS5A, proteine ​​NS5B) replica sono stati trattati con 3 micron HLF per 0, 2 o 24 ore. La colorazione doppia immunofluorescenza per le proteine ​​HLF e HCV NS5A è stata analizzata mediante microscopia in epifluorescenza. Questi risultati indicano che l&#39;HCV replicone subgenomico colorazione (come misurato con immunofluorescenza della HCV non strutturale proteina 5A) è stato ridotto in cellule trattate con HLF. Figura 5 </s<html> Trong&gt; rivela una riduzione qualitativa di circa il 50% in HCV intensità di colorazione dopo 24 ore di trattamento HLF. Questa è un&#39;osservazione interessante come questo sistema replicone subgenomico manca HCV E1 ed E2 glicoproteine, che sono i bersagli farmacologici comunemente accettati HLF. In concomitanza con il venir meno di HCV colorazione, l&#39;accumulo di HLF ha cominciato ad essere rilevato dopo 2 ore, e forte accumulo HLF è stata osservata dopo 24 ore. <img alt="Figura 1" src="/files/ftp_upload/53053/53053fig1.jpg" /> Figura 1. Principio di epifluorescenza. In questo tipo di microscopia, filtri di eccitazione selezionare la lunghezza d&#39;onda specifica che eccita il campione (tissus o celle ospitano anticorpi fluorescenti marcati). Inoltre, filtri di emissione sono scelti per abbinare le caratteristiche di emissione spettrali del fluoroforo utilizzato per etichettare il campione.carico / 53053 / 53053fig1large.jpg &quot;target =&quot; _ blank &quot;&gt; Clicca qui per vedere una versione più grande di questa figura. <img alt="Figura 2" src="/files/ftp_upload/53053/53053fig2.jpg" /> Figura 2. Principio di microscopia confocale. Questo tipo di microscopia utilizza un pinhole emissione di eliminare il segnale fuori fuoco luce. Dal momento che solo la luce prodotta da fluorescenza molto vicino al piano focale può essere rilevato, la risoluzione ottica dell&#39;immagine è molto migliore di quella di microscopi epifluorescenza. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53053/53053fig2large.jpg" target="_blank">Clicca qui per vedere una versione più grande di questa figura.</a> <img alt="Figura 3" src="/files/ftp_upload/53053/53053fig3.jpg" /> Figura 3. Schema di immunofluorescenza indiretta e diretta. Immunofluorescenza diretta coinvolgesolo tipo di anticorpo marcato con un fluorocromo, che è specifico per la molecola di interesse (anticorpo primario). Al contrario, immunofluorescenza indiretta coinvolge anticorpi secondari marcati con fluorocromi che sono specifici per la specie dell&#39;anticorpo primario senza etichetta. Più di un anticorpo secondario può allegare a ogni anticorpo primario che aumenta il segnale di fluorescenza. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53053/53053fig3large.jpg" target="_blank">Cliccate qui per vedere una versione più grande di questa figura.</a> <img alt="Figura 4" src="/files/ftp_upload/53053/53053fig4.jpg" /> Figura 4. epatocellulare assorbimento di esogeni Huh-7 cellule HLF. Sono state trattate con 0 pM o 3 micron HLF per 24 ore, lavato due volte, sottoposto ad un trattamento tripsina 5 minuti, ha permesso di ri-aderire per 18 ore e sottoposto a immunofluorescenza. I nuclei erano macchiati wesimo DAPI (blu canale), membrane plasmatiche sono stati etichettati con WGA (canale verde) e HLF era macchiato di un anticorpo anti-HLF (canale rosso). Le immagini sono state acquisite al microscopio confocale a un ingrandimento originale di 60x. Le sezioni ottiche orizzontali rappresentano la metà spessore delle celle. Barra della scala, 10 micron. Modificato da (11). <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53053/53053fig4large.jpg" target="_blank">Cliccate qui per vedere una versione più grande di questa figura.</a> <img alt="Figure5" src="/files/ftp_upload/53053/53053fig5.jpg" /> Figura 5. trattamento HLF riduce HCV colorazione. Huh-7 cellule di supporto replicazione di HCV replicone subgenomico sono stati trattati con 3 micron HLF per 0, 2, o 24 ore e sottoposti a immunofluorescenza. I nuclei sono stati colorati con DAPI (blu canale), l&#39;HCV è stato rivelato da un anticorpo anti-NS5A (canale rosso) e HLF era macchiato di un anti-HLF unntibody (canale verde). Le immagini sono state acquisite da epifluorescenza a un ingrandimento originale di 60x. Barra della scala, 10 micron. Modificato da (11). <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53053/53053fig5large.jpg" target="_blank">Cliccate qui per vedere una versione più grande di questa figura.</a>

Watch this Scientific Journal Video about Immunofluorescence to Monitor the Cellular Uptake of Human Lactoferrin and its Associated Antiviral Activity Against the Hepatitis C Virus at JoVE.com

Immunofluorescence to Monitor the Cellular Uptake of Human Lactoferrin and its Associated Antiviral Activity Against the Hepatitis C Virus

The human lactoferrin (hLF) is a component of the immune system. In this study, immunofluorescence assays are used to demonstrate both the hepatocellular uptake of hLF and a qualitative reduction in the hepatitis C virus replication upon treatment with hLF.

Immunofluorescenza per Monitorare il Cellular assorbimento di Lattoferrina umana e la sua associata attività antivirale contro il virus dell&#39;epatite C

Immunofluorescence is a laboratory technique commonly used to study many aspects of biology. It is typically used to visualize the distribution and/or ...

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13.3K Views.  Université de Sherbrooke. The human lactoferrin (hLF) is a component of the immune system. In this study, immunofluorescence assays are used to demonstrate both the hepatocellular uptake of hLF and a qualitative reduction in the hepatitis C virus replication upon treatment with hLF.

Video: Immunofluorescence to Monitor the Cellular Uptake of Human Lactoferrin and its Associated Antiviral Activity Against the Hepatitis C Virus

High-throughput Detection Method for Influenza Virus

Influenza virus is a respiratory pathogen that causes a high degree of morbidity and mortality every year in multiple parts of the world. Therefore, precise diagnosis of the infecting strain and rapid high-throughput screening of vast numbers of clinical samples is paramount to control the spread of pandemic infections. Current clinical diagnoses of influenza infections are based on serologic testing, polymerase chain reaction, direct specimen immunofluorescence and cell culture 1,2.
Here, we report the development of a novel diagnostic technique used to detect live influenza viruses. We used the mouse-adapted human A/PR/8/34 (PR8, H1N1) virus 3 to test the efficacy of this technique using MDCK cells 4. MDCK cells (104 or 5 x 103 per well) were cultured in 96- or 384-well plates, infected with PR8 and viral proteins were detected using anti-M2 followed by an IR dye-conjugated secondary antibody. M2 5 and hemagglutinin 1 are two major marker proteins used in many different diagnostic assays. Employing IR-dye-conjugated secondary antibodies minimized the autofluorescence associated with other fluorescent dyes. The use of anti-M2 antibody allowed us to use the antigen-specific fluorescence intensity as a direct metric of viral quantity. To enumerate the fluorescence intensity, we used the LI-COR Odyssey-based IR scanner. This system uses two channel laser-based IR detections to identify fluorophores and differentiate them from background noise. The first channel excites at 680 nm and emits at 700 nm to help quantify the background. The second channel detects fluorophores that excite at 780 nm and emit at 800 nm. Scanning of PR8-infected MDCK cells in the IR scanner indicated a viral titer-dependent bright fluorescence. A positive correlation of fluorescence intensity to virus titer starting from 102-105 PFU could be consistently observed. Minimal but detectable positivity consistently seen with 102-103 PFU PR8 viral titers demonstrated the high sensitivity of the near-IR dyes. The signal-to-noise ratio was determined by comparing the mock-infected or isotype antibody-treated MDCK cells.
Using the fluorescence intensities from 96- or 384-well plate formats, we constructed standard titration curves. In these calculations, the first variable is the viral titer while the second variable is the fluorescence intensity. Therefore, we used the exponential distribution to generate a curve-fit to determine the polynomial relationship between the viral titers and fluorescence intensities. Collectively, we conclude that IR dye-based protein detection system can help diagnose infecting viral strains and precisely enumerate the titer of the infecting pathogens.

This method describes the use of Infrared dye based imaging system for detection of H1N1 in bronchioalveolar lavage (BAL) fluid of infected mice at a high sensitivity. This methodology can be performed in a 96- or 384-well plate, requires &lt;10 &mu;l volume of test material and has the potential for concurrent screening of multiple pathogens.

High-throughput Metodo di rilevazione per il virus dell&#39;influenza

Influenza virus is a respiratory pathogen that causes a high degree of morbidity and mortality every year in multiple parts of the world. Therefore, ...

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Immunologia e infezioni

Two Methods of Heterokaryon Formation to Discover HCV Restriction Factors

Hepatitis C virus (HCV) is a hepatotropic virus with a host-range restricted to humans and chimpanzees. Although HCV RNA replication has been observed in human non-hepatic and murine cell lines, the efficiency was very low and required long-term selection procedures using HCV replicon constructs expressing dominant antibiotic-selectable markers1-5. HCV in vitro research is therefore limited to human hepatoma cell lines permissive for virus entry and completion of the viral life cycle. Due to HCVs narrow species tropism, there is no immunocompetent small animal model available that sustains the complete HCV replication cycle 6-8. Inefficient replication of HCV in non-human cells e.g. of mouse origin is likely due to lack of genetic incompatibility of essential host dependency factors and/or expression of restriction factors.
We investigated whether HCV propagation is suppressed by dominant restriction factors in either human cell lines derived from non-hepatic tissues or in mouse liver cell lines. To this end, we developed two independent conditional trans-complementation methods relying on somatic cell fusion. In both cases, completion of the viral replication cycle is only possible in the heterokaryons. Consequently, successful trans-complementation, which is determined by measuring de novo production of infectious viral progeny, indicates absence of dominant restrictions.
Specifically, subgenomic HCV replicons carrying a luciferase transgene were transfected into highly permissive human hepatoma cells (Huh-7.5 cells). Subsequently, these cells were co-cultured and fused to various human and murine cells expressing HCV structural proteins core, envelope 1 and 2 (E1, E2) and accessory proteins p7 and NS2. Provided that cell fusion was initiated by treatment with polyethylene-glycol (PEG), the culture released infectious viral particles which infected na&iuml;ve cells in a receptor-dependent fashion.
To assess the influence of dominant restrictions on the complete viral life cycle including cell entry, RNA translation, replication and virus assembly, we took advantage of a human liver cell line (Huh-7 Lunet N cells 9) which lacks endogenous expression of CD81, an essential entry factor of HCV. In the absence of ectopically expressed CD81, these cells are essentially refractory to HCV infection 10 . Importantly, when co-cultured and fused with cells that express human CD81 but lack at least another crucial cell entry factor (i.e. SR-BI, CLDN1, OCLN), only the resulting heterokaryons display the complete set of HCV entry factors requisite for infection. Therefore, to analyze if dominant restriction factors suppress completion of the HCV replication cycle, we fused Lunet N cells with various cells from human and mouse origin which fulfill the above mentioned criteria. When co-cultured cells were transfected with a highly fusogenic viral envelope protein mutant of the prototype foamy virus (PFV11) and subsequently challenged with infectious HCV particles (HCVcc), de novo production of infectious virus was observed. This indicates that HCV successfully completed its replication cycle in heterokaryons thus ruling out expression of dominant restriction factors in these cell lines. These novel conditional trans-complementation methods will be useful to screen a large panel of cell lines and primary cells for expression of HCV-specific dominant restriction factors.

We describe two methods for conditional trans-complementation of hepatitis C virus (HCV) assembly and the completion of the full viral life cycle, which rely on heterokaryon formation. These techniques are suitable to screen for cell lines that express dominant restriction factors, which preclude production of infectious HCV progeny.

Due metodi di Formazione Heterokaryon per scoprire fattori di restrizione HCV

Hepatitis C virus (HCV) is a hepatotropic virus with a host-range restricted to humans and chimpanzees. Although HCV RNA replication has been observed in ...

A Protocol for Analyzing Hepatitis C Virus Replication

Hepatitis C Virus (HCV) affects 3% of the world&#8217;s population and causes serious liver ailments including&#160;chronic hepatitis, cirrhosis, and hepatocellular carcinoma. HCV is an enveloped RNA virus belonging to the family Flaviviridae. Current treatment is not fully effective and causes adverse side effects. There is no HCV vaccine available. Thus, continued effort is required for developing a vaccine and better therapy. An HCV cell culture system is critical for studying various stages of HCV growth including viral entry, genome replication, packaging, and egress. In the current procedure presented, we used a wild-type intragenotype 2a chimeric virus, FNX-HCV, and a recombinant FNX-Rluc virus carrying a Renilla luciferase reporter gene to study the virus replication. A human hepatoma cell line (Huh-7 based) was used for transfection of in vitro transcribed HCV genomic RNAs. Cell-free culture supernatants, protein lysates and total RNA were harvested at various time points post-transfection to assess HCV growth. HCV genome replication status was evaluated by quantitative RT-PCR and visualizing the presence of HCV double-stranded RNA. The HCV protein expression was verified by Western blot and immunofluorescence assays using antibodies specific for HCV NS3 and NS5A proteins. HCV RNA transfected cells released infectious particles into culture supernatant and the viral titer was measured. Luciferase assays were utilized to assess the replication level and infectivity of reporter HCV. In conclusion, we present various virological assays for characterizing different stages of the HCV replication cycle.

Hepatitis C Virus (HCV) is a major human pathogen that causes liver disorders, including cirrhosis and cancer. An HCV infectious cell culture system is essential for understanding the molecular mechanism of HCV replication and developing new therapeutic approaches. Here we describe a protocol to investigate various stages of the HCV replication cycle.

Un protocollo per l&#39;analisi dell&#39;epatite C replicazione del virus

Hepatitis C Virus (HCV) affects 3% of the world&#8217;s population and causes serious liver ailments including&#160;chronic hepatitis, cirrhosis, and ...

A Method to Assess Fc-mediated Effector Functions Induced by Influenza Hemagglutinin Specific Antibodies

Antibodies play a crucial role in coupling the innate and adaptive immune responses against viral pathogens through their antigen binding domains and Fc-regions. Here, we describe how to measure the activation of Fc effector functions by monoclonal antibodies targeting the influenza virus hemagglutinin with the use of a genetically engineered Jurkat cell line expressing an activating type 1 Fc-Fc&#947;R. Using this method, the contribution of specific Fc-Fc&#947;R interactions conferred by immunoglobulins can be determined using an in vitro assay.

We describe a method to measure the activation of Fc-mediated effector functions by antibodies that target the influenza virus hemagglutinin. This assay can also be adapted to assess the ability of monoclonal antibodies or polyclonal sera targeting other viral surface glycoproteins to induce Fc-mediated immunity.

Un metodo per valutare le funzioni effettrici Fc-mediata indotte dagli anticorpi specifici di Hemagglutinin di riossidazione

Antibodies play a crucial role in coupling the innate and adaptive immune responses against viral pathogens through their antigen binding domains and ...

Detection of Viral RNA by Fluorescence in situ Hybridization (FISH)

Viruses that infect cells elicit specific changes to normal cell functions which serve to divert energy and resources for viral replication. Many aspects of host cell function are commandeered by viruses, usually by the expression of viral gene products that recruit host cell proteins and machineries. Moreover, viruses engineer specific membrane organelles or tag on to mobile vesicles and motor proteins to target regions of the cell (during de novo infection, viruses co-opt molecular motor proteins to target the nucleus; later, during virus assembly, they will hijack cellular machineries that will help in the assembly of viruses). Less is understood on how viruses, in particular those with RNA genomes, coordinate the intracellular trafficking of both protein and RNA components and how they achieve assembly of infectious particles at specific loci in the cell. The study of RNA localization began in earlier work. Developing lower eukaryotic embryos and neuronal cells provided important biological information, and also underscored the importance of RNA localization in the programming of gene expression cascades. The study in other organisms and cell systems has yielded similar important information. Viruses are obligate parasites and must utilise their host cells to replicate. Thus, it is critical to understand how RNA viruses direct their RNA genomes from the nucleus, through the nuclear pore, through the cytoplasm and on to one of its final destinations, into progeny virus particles 1.
FISH serves as a useful tool to identify changes in steady-state localization of viral RNA. When combined with immunofluorescence (IF) analysis 22, FISH/IF co-analyses will provide information on the co-localization of proteins with the viral RNA3. This analysis therefore provides a good starting point to test for RNA-protein interactions by other biochemical or biophysical tests 4,5, since co-localization by itself is not enough evidence to be certain of an interaction. In studying viral RNA localization using a method like this, abundant information has been gained on both viral and cellular RNA trafficking events 6. For instance, HIV-1 produces RNA in the nucleus of infected cells but the RNA is only translated in the cytoplasm. When one key viral protein is missing (Rev) 7, FISH of the viral RNA has revealed that the block to viral replication is due to the retention of the HIV-1 genomic RNA in the nucleus 8. 
Here, we present the method for visual analysis of viral genomic RNA in situ. The method makes use of a labelled RNA probe. This probe is designed to be complementary to the viral genomic RNA. During the in vitro synthesis of the antisense RNA probe, the ribonucleotide that is modified with digoxigenin (DIG) is included in an in vitro transcription reaction. Once the probe has hybridized to the target mRNA in cells, subsequent antibody labelling steps (Figure 1) will reveal the localization of the mRNA as well as proteins of interest when performing FISH/IF.

Detection of Viral RNA by Fluorescence in situ Hybridization FISH

A fluorescence in situ hybridization (FISH) method was developed to visually detect viral genomic RNA using fluorescence microscopy. A probe is made with specificity to the viral RNA that can then be identified using a combination of hybridization and immunofluorescence techniques. This technique offers the advantage of identifying the localization of the viral RNA or DNA at steady-state, providing information on the control of intracellular virus trafficking events.

Rilevamento di RNA virale per fluorescenza<em&gt; In situ</em&gt; Hybridization (FISH)

Viruses  that infect cells elicit specific changes to normal cell functions which serve  to divert energy and resources for viral replication. Many ...

Biologia

A Luciferase-fluorescent Reporter Influenza Virus for Live Imaging and Quantification of Viral Infection

Influenza A viruses (IAVs) cause human respiratory disease that is associated with significant health and economic consequences. As with other viruses, studying IAV requires the use of laborious secondary approaches to detect the presence of the virus in infected cells and/or in animal models of infection. This limitation has been recently circumvented with the generation of recombinant IAVs expressing easily traceable fluorescent or bioluminescent (luciferase) reporter proteins. However, researchers have been forced to select fluorescent or luciferase reporter genes due to the restricted capacity of the IAV genome for including foreign sequences. To overcome this limitation, we have generated a recombinant replication-competent bi-reporter IAV (BIRFLU) stably expressing both a fluorescent and a luciferase reporter gene to easily track IAV infections in vitro and in vivo. To this end, the viral non-structural (NS) and hemagglutinin (HA) viral segments of influenza A/Puerto Rico/8/34 H1N1 (PR8) were modified to encode the fluorescent Venus and the bioluminescent Nanoluc luciferase proteins, respectively. Here, we describe the use of BIRFLU in a mouse model of IAV infection and the detection of both reporter genes using an in vivo imaging system. Notably, we have observed a good correlation between the expressions of both reporters and viral replication. The combination of cutting-edge techniques in molecular biology, animal research and imaging technologies, provides researchers the unique opportunity to use this tool for influenza research, including the study of virus-host interactions and dynamics of viral infections. Importantly, the feasibility to genetically alter the viral genome to express two foreign genes from different viral segments opens up opportunities to use this approach for: (i) the development of novel IAV vaccines, (ii) the generation of recombinant IAVs that can be used as vaccine vectors for the treatment of other human pathogen infections.

Influenza A viruses (IAVs) are contagious respiratory pathogens that cause annual epidemics and occasional pandemics. Here, we describe a protocol to track viral infections in vivo using a novel recombinant luciferase and fluorescence-expressing bi-reporter IAV (BIRFLU). This approach provides researchers with an excellent tool to study IAV in vivo.

Un virus fluorescente di Luciferasi-fluorescente Reporter per l'imaging dal vivo e la quantificazione dell'infezione virale

Influenza A viruses (IAVs) cause human respiratory disease that is associated with significant health and economic consequences. As with other viruses, ...

Single-Cell Multiplexed Fluorescence Imaging to Visualize Viral Nucleic Acids and Proteins and Monitor HIV, HTLV, HBV, HCV, Zika Virus, and Influenza Infection

Capturing the dynamic replication and assembly processes of viruses has been hindered by the lack of robust in situ hybridization (ISH) technologies that enable sensitive and simultaneous labeling of viral nucleic acid and protein. Conventional DNA fluorescence in situ hybridization (FISH) methods are often not compatible with immunostaining. We have therefore developed an imaging approach, MICDDRP (multiplex immunofluorescent cell-based detection of DNA, RNA and protein), which enables simultaneous single-cell visualization of DNA, RNA, and protein. Compared to conventional DNA FISH, MICDDRP utilizes branched DNA (bDNA) ISH technology, which dramatically improves oligonucleotide probe sensitivity and detection. Small modifications of MICDDRP enable imaging of viral proteins concomitantly with nucleic acids (RNA or DNA) of different strandedness. We have applied these protocols to study the life cycles of multiple viral pathogens, including human immunodeficiency virus (HIV)-1, human T-lymphotropic virus (HTLV)-1, hepatitis B virus (HBV), hepatitis C virus (HCV), Zika virus (ZKV), and influenza A virus (IAV). We demonstrated that we can efficiently label viral nucleic acids and proteins across a diverse range of viruses. These studies can provide us with improved mechanistic understanding of multiple viral systems, and in addition, serve as a template for application of multiplexed fluorescence imaging of DNA, RNA, and protein across a broad spectrum of cellular systems.

Presented here is a protocol for a fluorescence imaging approach, multiplex immunofluorescent cell-based detection of DNA, RNA, and protein (MICDDRP), a method capable of simultaneous fluorescence single-cell visualization of viral protein and nucleic acids of different type and strandedness. This approach can be applied to a diverse range of systems.

Imaging a fluorescenza multiplexata a singola cellula per visualizzare acidi nucleici e proteine virali e monitorare HIV, HTLV, HBV, HCV, virus Zika e infezione influenzale

Capturing the dynamic replication and assembly processes of viruses has been hindered by the lack of robust in situ hybridization (ISH) technologies that ...

Measuring the Activation of Fc-Mediated Effector Functions by HA Antibodies

Source: Bailey, M. J., et al. <a href="https://app-jove-com.remotexs.ntu.edu.sg/v/56256">A Method to Assess Fc-mediated Effector Functions Induced by Influenza Hemagglutinin Specific Antibodies.</a> J. Vis. Exp. (2018).
This video demonstrates an assay measuring the antibody-mediated activation of T cell effector functions. Adding an influenza virus hemagglutinin (HA)-specific monoclonal antibody to transfected mammalian cells expressing HA results in the formation of immune complexes with HA. Upon introducing engineered T cells expressing Fc receptors and a luciferase reporter, the T cells are activated by the antibody-HA complexes, and this activation is detected by adding a luciferase substrate and measuring the produced bioluminescence.

Misurazione dell'attivazione delle funzioni effettrici mediate da FC da parte degli anticorpi HA

1. Expression of Influenza Virus Hemagglutinin via Transfection

	Plate Human Embryonic Kidney (HEK 293T) cells at a density of 2 x 104 cells/well in a ...

JoVE Encyclopedia of Experiments

Immunology

Antibody-Based Technologies

Characterization and Functional Analysis

An Immunofluorescence Method to Measure the Neutralization of Human Cytomegalovirus

This video demonstrates an immunofluorescence assay designed to measure the neutralization of Human Cytomegalovirus (HCMV). Virus-specific antibodies are mixed with the virus and incubated with human fibroblast cells. The antibodies neutralize the virus, rendering it unable to infect the host cells, while non-neutralized viruses infect the cells. After incubation, the extent of neutralization is determined by quantifying the virus-infected cells using immunofluorescence.

Un metodo di immunofluorescenza per misurare la neutralizzazione del citomegalovirus umano

1. Preparation of target cells for infection

	Seed target cells the day prior to performing the experiment in a 96-well plate (the &#39;target&#39; ...

Immunodiagnostics

Imaging and Visualization Techniques

A Viral Attachment Assay for Screening of Antiviral Compounds

Source: Tai, C. et al., <a href="https://app-jove-com.remotexs.ntu.edu.sg/v/53124">Early Viral Entry Assays for the Identification and Evaluation of Antiviral Compounds.</a> J. Vis. Exp.&#160;(2015)
This video demonstrates a viral attachment assay for screening antiviral compounds. Using genetically modified hepatitis C virus and luciferase, it assesses inhibitory effects on viral attachment, as indicated by lower light intensity in co-treated wells compared to wells treated with the virus alone.

Un test di attacco virale per lo screening di composti antivirali

NOTE: Ensure that all procedures involving cell culture and virus infection are conducted in certified biosafety hoods that are appropriate for the ...

Immunofluorescenza per Monitorare il Cellular assorbimento di Lattoferrina umana e la sua associata attività antivirale contro il virus dell'epatite C

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High-throughput Metodo di rilevazione per il virus dell'influenza