4. As a precaution, keep the previous passage.
5. Cultivate the cells in the incubator on the orbital shaker at
28
C and a suitable shaking rate.
3.4.4 Freezing the Cells
1. Calculate the volume of the Spodoptera frugiperda cell suspension needed to obtain a total of 3.6 Â 10
8 cells.
2. Split up the volume into 50-mL centrifuge tubes.
3. Centrifuge for 5 min at 200 Â g.
4. Fill 8.1 mL of supernatant into a 50-mL centrifuge tube and
add 1.8 mL DMSO to it.
5. Aspirate the remaining supernatant and resuspend the cells in
8.1 mL fresh medium.
6. Add the cell suspension to the prepared medium with DMSO
(see Note 14).
7. Fill 1 mL of the prepared cell suspension in each cryo-vial.
8. Transfer the 18 cryo-vials into the freezing container and freeze
at a rate of À1
C/min at À80
C for at least 4 h.
9. For long-term storage, transfer vials to the liquid nitrogen
freezing tank (À140
C).
10. To verify a successful freezing process, rethaw one vial
(described in Subheading 3.4.1). Observe cell growth and
viability. If a viability of >90% and a doubling time between
20 and 35 h can be observed 3–4 passages after rethawing,
successful freezing and rethawing is indicated.
3.5 Production
of the P1 Virus Stock
and Amplification
This part of the chapter describes the generation of a P1 virus stock
by a transfection of Sf-9 insect cells and the multiplication of the P1
virus stock to obtain the P2 virus stock.
3.5.1 Transfection
of Sf-9 Cells
for the Generation of a P1
Baculovirus Stock
1. Add 2 mL of Sf-9 cell suspension with a cell density of
0.3 Â 10
6 cells/mL to each well of a 6-well plate and incubate
at 28
C overnight.
2. Analyze the cell morphology with the microscope before infection. The cells should look round and not granulated. Further
no other particles (e.g., bacterial or fungal contamination)
should be visible.
3. Prewarm transfection reagent and vortex it.
4. Defrost the bacmid DNA (see Note 15).
5. Transfer 250 μL Grace’s Insect Medium into a 1.5 mL
centrifuge tube.
6. Add 2.5 μg bacmid DNA to the Grace’s Insect Medium.
7. Mix the bacmid DNA by gently inverting the centrifuge tube
10 times.
Antigen Production in Insect Cells
109
5. Cultivate the cells in the incubator on the orbital shaker at
28
C and a suitable shaking rate.
3.4.4 Freezing the Cells
1. Calculate the volume of the Spodoptera frugiperda cell suspension needed to obtain a total of 3.6 Â 10
8 cells.
2. Split up the volume into 50-mL centrifuge tubes.
3. Centrifuge for 5 min at 200 Â g.
4. Fill 8.1 mL of supernatant into a 50-mL centrifuge tube and
add 1.8 mL DMSO to it.
5. Aspirate the remaining supernatant and resuspend the cells in
8.1 mL fresh medium.
6. Add the cell suspension to the prepared medium with DMSO
(see Note 14).
7. Fill 1 mL of the prepared cell suspension in each cryo-vial.
8. Transfer the 18 cryo-vials into the freezing container and freeze
at a rate of À1
C/min at À80
C for at least 4 h.
9. For long-term storage, transfer vials to the liquid nitrogen
freezing tank (À140
C).
10. To verify a successful freezing process, rethaw one vial
(described in Subheading 3.4.1). Observe cell growth and
viability. If a viability of >90% and a doubling time between
20 and 35 h can be observed 3–4 passages after rethawing,
successful freezing and rethawing is indicated.
3.5 Production
of the P1 Virus Stock
and Amplification
This part of the chapter describes the generation of a P1 virus stock
by a transfection of Sf-9 insect cells and the multiplication of the P1
virus stock to obtain the P2 virus stock.
3.5.1 Transfection
of Sf-9 Cells
for the Generation of a P1
Baculovirus Stock
1. Add 2 mL of Sf-9 cell suspension with a cell density of
0.3 Â 10
6 cells/mL to each well of a 6-well plate and incubate
at 28
C overnight.
2. Analyze the cell morphology with the microscope before infection. The cells should look round and not granulated. Further
no other particles (e.g., bacterial or fungal contamination)
should be visible.
3. Prewarm transfection reagent and vortex it.
4. Defrost the bacmid DNA (see Note 15).
5. Transfer 250 μL Grace’s Insect Medium into a 1.5 mL
centrifuge tube.
6. Add 2.5 μg bacmid DNA to the Grace’s Insect Medium.
7. Mix the bacmid DNA by gently inverting the centrifuge tube
10 times.
Antigen Production in Insect Cells
109