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Baculovirus amplification in sf9 cells.

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Baculovirus amplification in sf9 cells.

Postby polinazzz » Mon Jun 27, 2011 5:23 pm

I have some doubts regarding virus amplification time. I have found instructions for working with sf9 cells and it is written there that incubation after cells infection with virus during amplification should be 72 hours (3days), because after that virus titer will decrease. But guys in our lab do 5 days incubation or sometimes even 6, they say that like that they get stronger virus.
If you have experience with insect cells, I would be grateful for some advise.
Thank you!
p.s. I do not measure virus titer, just take 2ml of virus for 10 million cells.
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Postby protold » Tue Jun 28, 2011 5:39 am

Hola in my experience 4 days is the ideal time to colect virus passages to get titers of 10e8 to 10e9 pfu/ml. you have to suposse the first passages titers and see light infection symptoms when you harvest. for exemople you have to suposse a 10e4 for the titer of a transfection supernatant of 4 days ,with it inoculate a p1 at a MOI around 0.2-0.5, after 4 days suposse a titer of 10e8(seeing light infection and less if you don´t see ) prepare a p2 in the same way in a B75flask with 1.5x10e7 cells, and after 4 days prepare inoculum for p3. at MOI0.2, after four days collect your p3(This is able to do repeated infections for protein production) and it would be better if you titer it because you will know what is the best MOI, but some people infect with some inoculum and harvest when the infection symptoms are very strong.the most accurate and cheap method to titer is by end-point dilution and there are in some protocols pages method descriptions and excell tools to calculate the titer. You dont say in wich passage you are I suposse that you have a p2 and want to prepare p3, or you want inoculate for protein production?. Well I will continue looking the forum if you need any aid. Buena suerte
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