Preparing Cells to Analyze Intracellular Uptake and Activity of an Antiviral Agent

Begin with human hepatoma cells cultured on coverslips in a multi-well plate.

These cells contain the hepatitis C virus genome in the cytoplasm, which produces viral proteins essential for viral genome replication.

In the control well, maintain the cells in culture medium alone, while in the test well, add lactoferrin, an antiviral agent.

Incubate the plate to allow lactoferrin to enter the cells through endocytosis and accumulate in the cytoplasm.

Inside the cytoplasm, lactoferrin disrupts the viral proteins, halting further viral genome replication.

In the control well, the viral genome replicates into negative-sense RNA intermediates, which are then used to produce positive-sense RNA copies that serve as templates for viral protein synthesis.

After treatment, wash the cells with a buffer to remove unbound lactoferrin.

Add a fixative solution with sucrose. Incubate to fix and stabilize the cells.

The cells are now ready for the analysis of intracellular uptake and antiviral activity of the treatment agent.

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-418. 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 0, 2, 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 Whatman 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