38. Day 7: Add 1 mL of each saturated overnight culture to 0.5 mL
of 50% glycerol in a 2-mL cryovial and gently mix. Freeze the
glycerol stock tube at À80
C. The stock is now stable for
years, as long as it is kept at À80
C.
39. Extract and purify the bacmid DNA from the remaining 5 mL
of each culture using a QIAquick spin miniprep kit, according
to the manufacturer’s instructions. Measure the DNA concentration using a NanoDrop spectrophotometer. The recombinant bacmid DNA is now ready for transfection into insect cells
to generate the baculovirus.
40. Take an aliquot of a properly maintained Sf9 stock culture,
count cells, and determine their viability (see Note 7).
41. Seed 1 Â 10
6 of Sf9 cells in 2 mL of PSFM-J1/FBS-PS
medium per well of six-well plate.
42. Incubate the cells at 27
C until they attach (~15 min).
43. While waiting for the cells to attach, add 6 μL of FuGENE
transfection reagent to 140 μL of 1Â Grace’s medium in a 1.5mL centrifuge tube for each transfection. Add 1 μg of bacmid
DNA to the FuGENE/1Â Grace media mixture, incubate for
15 min at 27
C, and then add 1Â Grace’s medium to make a
total volume of 500 μL.
44. Remove 2 mL of the supernatant from the seeded cells and
replace with 500 μL of the FuGENE/bacmid DNA mixture
dropwise onto the cells. Ensure even dispersal.
45. Incubate the cells for 4 h at 27
C, aspirate the transfectantDNA suspension, add 2 mL of fresh PSFM-J1/FBS-PS
medium, and return to 27
C (make sure to put the plates in
a sealable plastic bag to prevent strong evaporation of the
reagent/medium).
46. Incubate the cells for 96 h at 27
C.
47. Day 11: Collect the supernatant containing P0 virus (~2 mL
from each well), and filter the medium containing P0 virus into
a 2-mL centrifuge tube using a 3-mL syringe fitted with a small
0.2-μm filter. This virus is a stock of P0 virus that should be
stored at 4
C, protected from light.
48. Prepare a 125-mL Erlenmeyer flask (Vent filter cap/Baffle
bottom) containing 50 mL of Sf9 cells in the PSFM-J1/FBSPS medium at a density of 1.5 Â 10
6 cells/mL in the exponential growth phase.
49. Infect with 500 μL of P0 virus.
50. Incubate the cells for 96 h at 27
C with agitation (120 rpm).
51. Day 15: Transfer the suspension into a 50-mL tube, and centrifuge for 10 min at 2380 Â g at 4
C.
52. Collect the supernatant into a fresh 50-mL tube.
96
Norimichi Nomura et al.
of 50% glycerol in a 2-mL cryovial and gently mix. Freeze the
glycerol stock tube at À80
C. The stock is now stable for
years, as long as it is kept at À80
C.
39. Extract and purify the bacmid DNA from the remaining 5 mL
of each culture using a QIAquick spin miniprep kit, according
to the manufacturer’s instructions. Measure the DNA concentration using a NanoDrop spectrophotometer. The recombinant bacmid DNA is now ready for transfection into insect cells
to generate the baculovirus.
40. Take an aliquot of a properly maintained Sf9 stock culture,
count cells, and determine their viability (see Note 7).
41. Seed 1 Â 10
6 of Sf9 cells in 2 mL of PSFM-J1/FBS-PS
medium per well of six-well plate.
42. Incubate the cells at 27
C until they attach (~15 min).
43. While waiting for the cells to attach, add 6 μL of FuGENE
transfection reagent to 140 μL of 1Â Grace’s medium in a 1.5mL centrifuge tube for each transfection. Add 1 μg of bacmid
DNA to the FuGENE/1Â Grace media mixture, incubate for
15 min at 27
C, and then add 1Â Grace’s medium to make a
total volume of 500 μL.
44. Remove 2 mL of the supernatant from the seeded cells and
replace with 500 μL of the FuGENE/bacmid DNA mixture
dropwise onto the cells. Ensure even dispersal.
45. Incubate the cells for 4 h at 27
C, aspirate the transfectantDNA suspension, add 2 mL of fresh PSFM-J1/FBS-PS
medium, and return to 27
C (make sure to put the plates in
a sealable plastic bag to prevent strong evaporation of the
reagent/medium).
46. Incubate the cells for 96 h at 27
C.
47. Day 11: Collect the supernatant containing P0 virus (~2 mL
from each well), and filter the medium containing P0 virus into
a 2-mL centrifuge tube using a 3-mL syringe fitted with a small
0.2-μm filter. This virus is a stock of P0 virus that should be
stored at 4
C, protected from light.
48. Prepare a 125-mL Erlenmeyer flask (Vent filter cap/Baffle
bottom) containing 50 mL of Sf9 cells in the PSFM-J1/FBSPS medium at a density of 1.5 Â 10
6 cells/mL in the exponential growth phase.
49. Infect with 500 μL of P0 virus.
50. Incubate the cells for 96 h at 27
C with agitation (120 rpm).
51. Day 15: Transfer the suspension into a 50-mL tube, and centrifuge for 10 min at 2380 Â g at 4
C.
52. Collect the supernatant into a fresh 50-mL tube.
96
Norimichi Nomura et al.