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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.
1. Tissue Culture Incubator
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.
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.
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