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

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

Immunofluorescence is a laboratory technique commonly used to study many aspects of biology. It is typically used to visualize the distribution and/or localization of a target molecule in cells and tissues. Immunofluorescence relies on the specificity of fluorescent-labelled antibodies against their corresponding antigens within a cell. Both direct and indirect immunofluorescence approaches can be used which rely on the use of antibodies linked with a fluorochrome. Direct immunofluorescence is less frequently used because it provides lower signal, involves higher cost and less flexibility. In contrast, indirect immunofluorescence is more commonly used because of its high sensitivity and provides an amplified signal since more than one secondary antibody can attach to each primary antibody. In this manuscript, both epifluorescence microscopy and confocal microscopy were used to monitor the internalization of human lactoferrin, an important component of the immune system, into hepatic cells. Moreover, we monitored the inhibitory potential of hLF on the intracellular replication of the Hepatitis C virus using immunofluorescence. Both the advantages and disadvantages associated with these approaches are discussed.

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)....

Protocol

1. Cells Preparation and Treatment

1. In a 24-well plate, put a glass cover at the bottom of the wells. Wash each well with Phosphate Buffered Saline (PBS).
2. Seed Huh-7 cells supporting HCV subgenomic replicon in each well to a density of 5 × 10⁴ cells/well. If there is no treatment to do, the cells can be seeded at a higher density. The culture medium is Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 10% Foetal Bovine Serum (FBS), 2mM L-glutamine, 1 mM sodium pyruvate and 250 mg/ml G-418 (to maintain the replicon).
3. Incubate at 37 °C and 5 % CO_2. The next day, treat the cells w....

Results

The ability of the hepatic Huh-7 cell line to internalize exogenously administrated hLF was monitored using immunofluorescence on a confocal miscroscope. hLF was added to the culture media and the internalization was allowed for 24 hr, after which, extracellular hLF was washed with PBS and residual membrane bound hLF was degraded with a 5 min trypsin treatment (1 ml). Cells were reseeded and allowed to re-adhere for 18 hr before immunofluorescence staining. In order to outline the cytoplasmic limits, the cells membranes .......

Discussion

The HCV epidemic remains a global threat, with 80% of newly infected patients developing a chronic infection, putting them at risk of cirrhosis, liver failure or hepatocellular carcinoma. Direct-acting antiviral agents targeting HCV replication and maturation represent prime anti-HCV agents, as recently demonstrated by the regulatory approval of two NS3 N-terminus protease inhibitors (Boceprevir and Telaprevir). The hLF anti-HCV activity is currently believed to behave as a viral entry inhibitor, binding circulating viri.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
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