How To Make Baculovirus High-Titer Stocks

single use bioreactor
Baculovirus stocks have infectious titers that usually range between 6×106 and 3×108 pfu/mL. At Kempbio we have found that the best way to ensure that your working baculovirus titers are in the range of 1×108 pfu/mL is to infect Sf9 cells using a low MOI, (we use 0.02) and to incubate at 27°C for 3 days. It is also important to split your Sf9 cells to 5×105 cells per mL 18 – 24 hours before adding virus to make stock. Baculovirus stocks with good titer levels can be made in static flasks, shake flasks or stirred-tank bioreactors. We normally produce P0 stocks in T-25 flasks with 6 mL of serum-free medium and we produce most of our high-titer virus stocks in shake-flask culture unless we are running BacMam expressions at the 100-liter scale where we may be adding 10-liters of very fresh virus stock which we produce using a stirred-tank bioreactor. We titer our virus stocks using the ViroCyt Virus Counter but titers may also be determined by sending samples to Expression Systems (, running some of the commercially available immunoassay kits or performing a standard plaque assay (protocol on our website).

If you do not know the titer of the virus stock that you are trying to passage to make a high-titer virus stock you might want to try a series of small amplifications using a range of volumetric virus additions. For example, you could set up four, 250 mL shake flasks containing 50 mL of medium with Sf9 cells at 5×105 cells per mL. Incubate the flasks at 27°C on a shaker running at 80-100 rpm and add virus after 18-24 hours of incubation. The volumes of virus that we would recommend that you add separately to the four flasks is 0.01, 0.1, 0.5 and 1 mL. Incubate the flasks for 72h. at 27°C and obtain viable cell counts before you harvest. You are looking for a cell count of between 2 x106 and 4 x106 cells per mL and a viability of less than 70%. That should give you a virus stock with a titer somewhere around 1×108 pfu/mL. If the viability is high and the count is above 5×106 cells per mL the titer may be low. The titer will also be low if the cells growth arrested at 1×106 cells per mL and the viability is low (<50%). For test expressions you can use the same 50 mL in a 250 mL flask setup using Sf9 cells at a density of 1.5×106 cells per mL. If we know the titer of the virus that we are using we test express using an MOI of 0.1 and 1 and take samples at 48 and 72h. post-infection for western blot analysis. If you do not know the virus titer try running your test expressions using virus volumes of 0.2 and 2% V/V.