8. Incubate the transfection solution at room temperature for
15 min.
9. Dropwise add the transfection solution to the cell suspension
of each well.
10. Incubate the 6-well plates at 28
C for 72 h, without shaking.
11. Again, analyze the morphology of the insect cells under the
microscope and check the cells for cytopathic effects (see Note
16).
12. Gently transfer the cell suspension to sterile 15 mL centrifuge
tubes.
13. Centrifuge the tubes for 5 min at 1000 Â g.
14. Transfer the P1 virus stock-containing supernatant into new
15 mL centrifuge tubes that are impervious for light because of
the virus’s light sensitivity.
15. Store the centrifuge tubes at 4
C, protected from light.
3.5.2 Amplification
of the P1 Baculovirus Stock
to P2
1. Add 50 mL of Sf-9 cell suspension with a cell density of
1 Â 10
6 cells/mL to a baffled shake flask with a working
volume of 250 mL (see Note 17).
2. Add 2 mL of P1 virus stock to the cell suspension.
3. Wrap the flask in aluminum foil and incubate the cell suspension containing viruses at 80 rpm and 28
C for 72–96 h.
4. Determine the cell concentration and the viability directly after
infection, and continue sampling every 24 h to monitor the
progress of infection.
5. As soon as the viability of the Sf-9 cells drops below of 75–80%,
harvest the virus containing cell suspension (see Note 18).
6. Transfer the harvested cell suspension to sterile 50 mL centrifuge tubes and centrifuge them at 250 Â g for 10 min.
7. Transfer the supernatant into new 50 mL centrifuge tubes and
centrifuge the tubes at 3000 Â g for 10 min.
8. Again, transfer the P2 virus-containing supernatant to a fresh
50 mL centrifuge tubes (see Note 19).
9. Store the centrifuge tubes at 4
C protected from light.
3.6 Determination
of the Baculoviral Titer
This part of the chapter describes three different methods for virus
titration: the quantification using qPCR with SYBR Green (see
Note 20), the performance of a viral plaque assay (see Note 21),
and the determination of a TCID 50 (see Note 22).
3.6.1 Quantification
of Baculoviruses Using
qPCR with SYBR Green
The quantification of the baculoviral titer using qPCR with SYBR
Green is carried out according to the manufacturer’s specifications
by Thermo Fisher for the corresponding qPCR Kit [9].
110
Julie Harnischfeger et al.
15 min.
9. Dropwise add the transfection solution to the cell suspension
of each well.
10. Incubate the 6-well plates at 28
C for 72 h, without shaking.
11. Again, analyze the morphology of the insect cells under the
microscope and check the cells for cytopathic effects (see Note
16).
12. Gently transfer the cell suspension to sterile 15 mL centrifuge
tubes.
13. Centrifuge the tubes for 5 min at 1000 Â g.
14. Transfer the P1 virus stock-containing supernatant into new
15 mL centrifuge tubes that are impervious for light because of
the virus’s light sensitivity.
15. Store the centrifuge tubes at 4
C, protected from light.
3.5.2 Amplification
of the P1 Baculovirus Stock
to P2
1. Add 50 mL of Sf-9 cell suspension with a cell density of
1 Â 10
6 cells/mL to a baffled shake flask with a working
volume of 250 mL (see Note 17).
2. Add 2 mL of P1 virus stock to the cell suspension.
3. Wrap the flask in aluminum foil and incubate the cell suspension containing viruses at 80 rpm and 28
C for 72–96 h.
4. Determine the cell concentration and the viability directly after
infection, and continue sampling every 24 h to monitor the
progress of infection.
5. As soon as the viability of the Sf-9 cells drops below of 75–80%,
harvest the virus containing cell suspension (see Note 18).
6. Transfer the harvested cell suspension to sterile 50 mL centrifuge tubes and centrifuge them at 250 Â g for 10 min.
7. Transfer the supernatant into new 50 mL centrifuge tubes and
centrifuge the tubes at 3000 Â g for 10 min.
8. Again, transfer the P2 virus-containing supernatant to a fresh
50 mL centrifuge tubes (see Note 19).
9. Store the centrifuge tubes at 4
C protected from light.
3.6 Determination
of the Baculoviral Titer
This part of the chapter describes three different methods for virus
titration: the quantification using qPCR with SYBR Green (see
Note 20), the performance of a viral plaque assay (see Note 21),
and the determination of a TCID 50 (see Note 22).
3.6.1 Quantification
of Baculoviruses Using
qPCR with SYBR Green
The quantification of the baculoviral titer using qPCR with SYBR
Green is carried out according to the manufacturer’s specifications
by Thermo Fisher for the corresponding qPCR Kit [9].
110
Julie Harnischfeger et al.