Summary

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

Published: October 01, 2015
doi:

Summary

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.

Abstract

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'interno di una cella. Entrambi gli approcci diretti e indiretti immunofluorescenza possono essere utilizzati che si basano sull'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'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'epatite C mediante immunofluorescenza. Entrambi i vantaggi e gli svantaggi associati a questi approcci sono discussi.

Introduction

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).

Protocol

1. Le celle di preparazione e trattamento In una piastra a 24 pozzetti, mettere una copertura in vetro sul fondo dei pozzetti. Lavare ogni pozzetto con tampone fosfato (PBS). 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% sier…

Representative Results

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'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 deline…

Discussion

L'epidemia HCV rimane una minaccia globale, con l'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'attività anti-HCV HLF è attualmente crede di comportarsi co…

Disclosures

The authors have nothing to disclose.

Acknowledgements

This work was funded by both the Canadian Institutes of Health Research and Natural Sciences and the Engineering Research Council of Canada. M. Bisaillon is a Chercheur Boursier Senior from the Fonds de Recherche en Santé du Québec 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.

Materials

DMEM Wisent 319-005-CL
PAF BioShop PAR070.1 Flammable solid, skin irritant, lungs and eyes. 
PBS Wisent 311-425-CL Without Ca2+ & Mg2+
NGS Wisent 053-150
AlexaFluor 488-labeled anti-mouse Invitrogen A11017
AlexaFluor 568-labeled anti-rabbit Invitrogeb A21069
Wheat germ agglutinin Alexa Fluor 488 conjugate (WGA) Invitrogen W11261 Potentially mutagenic
Anti-NS5A rabbit Abcam ab2594
Anti-hLF mouse Abcam ab10110
SlowFade Invitrogen S36937
Hoechst stain Life Techn. H1399 Potentially mutagenic and carcinogenic
hLF Sigma L0520
Nikon Eclipse visible/epifluorescence Microscope Nikon TE2000-E
epifluorescence/confocal microscope Olympus FV1000

References

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Cite This Article
Allaire, A., Picard-Jean, F., Bisaillon, M. Immunofluorescence to Monitor the Cellular Uptake of Human Lactoferrin and its Associated Antiviral Activity Against the Hepatitis C Virus. J. Vis. Exp. (104), e53053, doi:10.3791/53053 (2015).

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