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

Resumo

Source: Allaire, A., et al. Immunofluorescence to Monitor the Cellular Uptake of Human Lactoferrin and its Associated Antiviral Activity Against the Hepatitis C Virus. J. Vis. Exp. (2015).

The video demonstrates the procedure for preparing cells to assess the intracellular uptake and activity of an antiviral agent against the hepatitis C virus.

Protocolo

1. Cell 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 hepatitis C virus (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% Fetal 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% CO₂. The next day, treat the cells with 3 µM of human lactoferrin (hLF) purified from human milk.

2. Paraformaldehyde/Sucrose Preparation (12% PFA and 12% sucrose)

1. Put 500 ml of PBS in a beaker and heat it between 20 °C and 30 °C. Dissolve 60 g of sucrose in the PBS. Dissolve 60 g of paraformaldehyde (PFA) in the PBS/sucrose solution. Slowly add sodium hydroxide 2N (3 to 7 ml) until a clear solution is obtained.
2. Adjust pH to 7.4 with hydrochloric acid. Complete volume to 500 ml with PBS. Filter by gravity on a Whatman filter. Before use, dilute the solution with PBS to a final concentration of 4% PFA and 4% sucrose (Storage 4 °C for 1 week maximum).
3. At the desired time (0 hr, 2 hr, or 24 hr of treatment), wash cells twice (1 min) with PBS and fix with PBS/4% PFA/4% sucrose for 20 min at room temperature. The cells are typically at 30-40% confluence, and 1.5 × 10⁵ cells are used.

Materiais

Lista de materiais utilizados neste artigo

Nome	Empresa	Número de catálogo	Comentários
Dulbecco’s Modified Eagle Medium (DMEM)	Wisent	319-005-CL
Paraformaldehyde (PFA)	BioShop	PAR070.1	Flammable solid, skin irritant, lungs and eyes
Phosphate-buffered saline (PBS)	Wisent	311-425-CL	Without calcium & magnesium ions
Human lactoferrin (hLF)	Sigma	L0520