How to Make High Titer Baculovirus Stocks

This protocol is based on a procedure posted on the Invitrogen Baculovirus User Forum.

1. Plate out 1.5x10^7 Sf9 cells in a 10cm tissue culture dish. Allow one hour for the cells to adhere.

2. Remove the medium and infect the cells at a multiplicity of infection around 0.1-0.5, in a total volume of 5 ml. Rock for one hour.

3. Add 10 ml medium and incubate 2-3 days at 28 degrees C.

4. Use a serological pipet to wash the cells off the dish, and place the cell suspension in a centrifuge tube. It reportedly may also help to pipet the cells up and down several times in the tube (I've never verified this, however).

5. Remove the cells by centrifugation for 15 minutes at 2000xg.

6. The resulting supernatant should contain budded virus at a concentration of approximately 5-10x10^8 pfu/ml.

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