[Bio101] Lentivirus Infection

Materials and Reagents

1.        6 cm tissue culture plates (Fiosher).

2.        Human or mouse cell line and appropriate growth media Reagents required for cell-based assay.

3.        Polybrene (Hexadimethrine bromide; Sigma H 9268 )

4.        Appropriated antibiotics for selection purpose.

Equipment

1.        Tissue Culture Incubator

Procedure

Note: Lentiviral infections should be optimized for each cell line and cell-based assay. For example, the following parameters should be tested before starting large-scale infections to determine the optimal conditions for a given experiment:

1)       Cell seeding density.

2)       Amount of lentivirus.

3)       Puromycin concentration.

4)       Timecourse.

1.        Seed cells at appropriate density in 6 mL in 6 cm plates.

1)       Adherent cells: seed 1 day prior to infection.

2)       Suspension cells: seed day of infection in media containing polybrene.

2.        Add virus to cells:

1)         (Adherent cells): Remove growth media and add fresh media containing polybrene. Alternatively, remove a portion of the growth media and supplement with media containing polybrene. Adjust volumes and polybrene concentration to achieve the correct final polybrene concentration (8ug/ml).

3.        Viral infection:

1)       Incubate cells overnight.

2)       Change media 24 hours post-infection. Remove media and replace with 6 mL fresh growth media. If antibiotics selection is desired, use fresh growth media containing antibiotics. Note: Puromycin concentration should be optimized     for each cell line; typical concentrations range from 2-5 μg/mL.

4.        Incubate cells, replacing growth media (with antibiotics, if desired) as needed every few days. Incubation periods are highly dependent on the post-infection assay.

Note: All lentiviral procedures should be carried out in accordance with biosafety requirements of the host institution.