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

The overall goal of the following experiment is to monitor the internalization of human lactoferrin into hepatic cells using both epi fluorescence and confocal microscopy, and to evaluate its inhibitory potential on the intracellular replication of the Hepatitis C virus or HCV. This is achieved by growing HUH seven cells, supporting the HCV sub genomic replicon. Next human lactoferrin is added to the growing cells, which allows the protein to enter the cells.

Then the cells are fixed and visualized using fluorescence microscopy. The results show that hepatocytes have the capacity to acquire HLF from their extracellular environment and the HCV sub genomic replicon staining is reduced in cells treated with HLF based on fluorescence microscopy. This method can help answer key question in the EB C virus field, such as the molecular mechanism by which the rine inhibit the replication of the virus.

To begin place a round cover glass in the bottom of each well of a 24 well plate and use PBS to wash the wells seed, HUH seven cells carrying the HCV Replicon at a density of five times 10 of the fourth cells per well. In DMEM supplemented with 10%FBS two millimolar, L glutamine, one millimolar sodium pyruvate, and 250 milligrams per milliliter. G four 18 teen incubate the cells at 37 degrees Celsius and 5%carbon dioxide overnight.

The following day, treat the cells with three micromolar, human lactoferrin or HLF purified from human milk and incubate for zero two or 24 hours to prepare para formaldehyde or PFA in sucrose at 350 milliliters of PBS into a beaker and heat it to between 20 and 30 degrees Celsius at 60 grams of sucrose and allow it to dissolve before dissolving 60 grams of PFA in the solution. Then slowly add three to seven milliliters of two normal sodium hydroxide until a clear solution is obtained. After using HCL to adjust the pH to 7.4, use PBS to bring the volume to 500 milliliters.

Then with watman paper, filter the solution by gravity before use. ADD PBS to dilute the solution to 4%PFA, and 4%sucrose at the desired time after HLF treatment. Use PBS to wash the cells twice for one minute each, and use the diluted PFA sucrose solution to fix the cells at room temperature for 20 minutes to carry out immunofluorescence use 0.15%Triton X 100 in PBS to perme.

Mobilize the fixed cells at room temperature for five minutes. After washing the cells with PBS, add 10%NGS in PBS and block for 20 minutes in 10%NGS dilute the primary antibody of interest and add to the cells. Incubate at room temperature for four hours or at four degrees Celsius overnight with 10%NGS.

Wash the cells three times for five minutes each and add a fluorescently labeled secondary antibody diluted one to 1000 in NGS Incubate in the dark at room temperature for one hour after washing the cells once with PBS for five minutes, apply DPI and incubate in the dark at room temperature for 15 minutes. Use PBS to wash the cells twice and use a slow fade mounting medium to mount the cells for microscopy. Refer to the text protocol for control samples.

Finally, to image the samples, use epi fluorescence or confocal microscopy with the appropriate filters and use an oil immersion lens to increase the numerical aperture of the lens. Following the addition of exogenous HLF to the hepatic H two H seven cell line, a significant amount of HLF was detected inside the cytoplasm of hepatocytes no HLF was observed inside the untreated cells. These data confirmed that hepatocytes have the capacity to acquire HLF from their extracellular environment.

To monitor the ability of HLF to inhibit the intracellular replication of H-C-V-H-U-H seven cells. Supporting the HCV sub genomic REPLICON were treated with three micromolar HLF for zero two or 24 hours. Immunofluorescence shows that HCV sub genomic replicon staining was reduced about 50%in cells treated with HLF.

After 24 hours concomitant with the weaker HCV signal detection of HLF accumulation began after two hours and strong HLF accumulation was observed. After 24 hours Following this procedure, additional method like protein ation can be performed in order to answer additional question like, does the lactoferrin protein interact with a specific HCV protein.