Contributed Tue Jan 25 19:34:54 PST 2000 by:
Robin Clark
Protemation Inc.
Benicia, CA USA

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.


  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.

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