How to Infect Mammalian Cell Cultures with Viruses

Biochemistry, Molecular Biology, and Cell Biology Protocols >> How to Infect Mammalian Cell Cultures with Viruses

1. Split cells the day before so that the next days the cells are at the desired confluency (40-50% for immunofluorescence studies). Split cells onto glass cover slips if fluorescence microscopy will be performed later.
2. Wash cells with PBS the next day.
3. Replace with new media containing 0.2% fetal bovine serum.
4. Add virus at the desired multiplicity of infection (MOI). An MOI of 1 should be adequate for most studies, but an MOI of approximately 0.01 to 100 has also been useful for various studies.
5. Let the virus bind to the surface of the cells for 30 minutes to 1 hour (maintain a constant binding time each time) at room temperature in order to synchronize viral entry. (4-degree incubation on ice may be necessary if the virus can enter the cell at room temperature.)
6. After 30 minutes to 1 hour of binding, remove the unbound virus in the medium, and replace with new medium containing 2% fetal bovine serum.
7. Place the cells into the 37-degree C incubator for the desired amount of time. For the cells to express new viral proteins, overnight incubation will usually allow enough time for the new proteins to be expressed.