6. Grace’s Insect Medium (e.g., Sigma-Aldrich, St. Louis,
Missouri, USA).
7. 6-well plates for suspension culture (e.g., Sarstedt, Nu ¨mbrecht,
Germany).
8. Sterile single-use pipette tips (e.g., serological pipettes, Sarstedt, Nu ¨mbrecht, Germany).
9. Pipette holder/rubber bulb (e.g., pipetus, Hirschmann Laborger€ ate, Eberstadt, Germany).
10. Microscope (e.g., Inverted Microscope Dmi1, Leica Camara,
Wetzlar, Germany).
11. Aspiration system (e.g., Vacusafe, Integra, Biebertal,
Germany).
12. Multifuge (e.g., Multifuge X1R, Thermo Fisher Scientific,
Waltham, MA, USA).
13. 15-mL centrifuge tubes, lightsafe (e.g., Sarstedt, Nu ¨mbrecht,
Germany).
14. Incubator (e.g., KB 115 E 3.1, Binder, Tuttlingen, Germany).
15. Centrifuge (e.g., 6-16 KS, Sigma-Aldrich, St. Louis,
Missouri, USA).
2.5.2 Materials
for Amplification of the P1
Baculovirus Stock to P2
1. Material for cell counting (see Subheading 2.4.2).
2. Spodoptera frugiperda cell suspension with a cell density of
1.0 Â 10
6 cells/mL (e.g., TriEx Sf-9, Merck, Darmstadt,
Germany).
3. Insect cell culture medium (e.g., Sf900 II, Thermo Fisher
Scientific, Waltham, MA, USA).
4. P1 baculovirus stock (2 mL) (e.g., AcMNPV-Orf2-PCV2; see
Note 6).
5. 250 mL baffled shake flask (e.g., Glasger€ atebau Ochs, Bovenden, Germany).
6. Sterile single-use pipette tips (e.g., serological pipettes, Sarstedt, Nu ¨mbrecht, Germany).
7. Pipette holder/rubber bulb (e.g., pipetus, Hirschmann Laborger€ ate, Eberstadt, Germany).
8. 50-mL centrifuge tubes, lightsafe (e.g., Sarstedt, Nu ¨mbrecht,
Germany).
9. Incubator (e.g., KB 115 E 3.1, Binder, Tuttlingen, Germany).
10. Centrifuge (e.g., 6-16 KS, Sigma-Aldrich, St. Louis,
Missouri, USA).
2.6 Determination
of the Baculovirus
Titer
In this part of the chapter, three alternatives for the determination
of the baculovirus titer are presented: the quantification using
qPCR with SYBR Green, the performance of a viral plaque assay,
and the determination the TCID 50 .
Antigen Production in Insect Cells
103