Protocols_CellCulture_Lentivirus infection

Lentiviral Infection

• The viral supernatant is aliquoted into tubes of 200-ul virus and should be kept frozen at -80C.

• After freezing, remember that each freeze-thaw will decrease viral titer so be careful and consistent.

• Day 1: 1x10^5^ cells per well were plated into a 12-well plate in the appropriate standard media with 8 ug/ml polybrene.

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• Put 100 ul viral supernatant per well and shake the cells gently to equally distribute the viruses. Viral supernatant can be put without waiting for cells to attach to the surface.

• Try to use viral supernatant immediately after thawing. Be gentle while pipetting the viral supernatant.

• Day 2: Change the medium into regular growth medium.

• Day 3: Add puromycin (0.5 ug/ml for HCT116 cells). After 3 days (or as soon as there are no surviving cells in the no-transduction control under puromycin), cells will be ready for analysis.