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 &#x....

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