Recovering Virus Stocks from Frozen Cell Pellets

If your recombinant baculovirus stocks have been lost or have "gone off", virus can usually be recovered from frozen pellets of baculovirus-infected insect cells. Virus in these pellets is stable for many years, especially if stored at -70 C or less.

Procedure:

1. Resuspend the frozen pellet in 10 volumes of insect cell growth medium supplemented with 5% FBS. Vortex briefly.
2. Centrifuge at low speed to remove cell debris (1-2000 x g for 5 min.).
3. Filter supernate with 0.45 micron sterile filter (not O.2 micron).
4. Infect insect cells by adding a volume of filtered virus to the cell cultures that is equivalent to 5% of the total media volume. Normally this is done using stationary cultures at about 5E5 cells/ml in a vessel such as a T-125 flask (10-12 ml total per flask) with media containing 2-5% FBS (FBS enhances virus stability).
5. Harvest virus stocks when all cells appear infected, or when they become confluent (3-5 days). Some toxicity, affecting 10-40% of the cells, usually occurs within 24 hours of infection. Re-passage the virus stock as necessary.