(e.g., Cytation3, BioTek, Winooski, Vermont, USA). For an
indication of the purity, the ratio of E 260 to E 280 can be
determined.
16. The infection of insect cells with baculoviral DNA usually leads
to structural changes and cytopathic effects [10, 11]. Therefore,
it is useful to morphologically analyze the host cells before and
after a viral transfection.
17. The inoculum concentration is calculated for a total culture
volume of 50 mL—however, only 48 mL culture volume is
required. The viral infection is carried out by adding the 2 mL
of P1 virus stock.
18. Virus harvesting should be performed before the insect cells
enter the stationary phase. Therefore, the viability of the insect
cells should be monitored. If the viability decreases too much,
viral surface proteins can be degraded by cell lysis-related host
cell proteins and, as a result, the infectivity of the virus
decreases.
19. Further measurements can be carried out with the supernatant,
e.g., concentration of amino acids, or glucose, or lactate.
20. To quantify the baculovirus using qPCR with SYBR Green, a
specific gene sequence of the bacmid DNA must be selected,
and corresponding primers need to be designed. In addition,
the copy number needs to be calculated using the following
equation:
Copy Number ¼
Amount ng
ð Þ Â 6:022 Â 10
23
Length bp
ð Þ Â 1 Â 10
9
 660
The Amount (ng) corresponds the amount of DNA.
6.022 Â 10
23 is the Avogadro’s constant, which indicates the
number of particles in 1 mol. The Length (bp) is the length of
the DNA in bp. 1 Â 10
9 represents the conversion factor for
ng, and 660 is the average mass of 1 bp double stranded DNA.
21. For performing a viral plaque assay, Sf-9 Insect Cells (e.g., Sf-9
Insect Cells Novagen, Merck, Darmstadt, Germany) are
required. For this purpose, the insect cells are cultivated semiadherently in T-bottles, as specified by the manufacturer.
22. The TCID 50 is determined, for example, by the cytopathic
effect of insect cells, or by a reporter gene that codes for a
fluorescent protein, such as the green fluorescent protein
(GFP).
23. Use a multichannel pipette for the virus dilutions 10
À0 to 10
À7
in order to process all samples simultaneously.
24. It is recommended, to attach one type of Luer lock adapter to
the tubes connected to the headplate (e.g., male Luer lock),
and the opposite adapter (e.g., female Luer lock) to all tubes
connected to bottles. This prevents confusion.
116
Julie Harnischfeger et al.
indication of the purity, the ratio of E 260 to E 280 can be
determined.
16. The infection of insect cells with baculoviral DNA usually leads
to structural changes and cytopathic effects [10, 11]. Therefore,
it is useful to morphologically analyze the host cells before and
after a viral transfection.
17. The inoculum concentration is calculated for a total culture
volume of 50 mL—however, only 48 mL culture volume is
required. The viral infection is carried out by adding the 2 mL
of P1 virus stock.
18. Virus harvesting should be performed before the insect cells
enter the stationary phase. Therefore, the viability of the insect
cells should be monitored. If the viability decreases too much,
viral surface proteins can be degraded by cell lysis-related host
cell proteins and, as a result, the infectivity of the virus
decreases.
19. Further measurements can be carried out with the supernatant,
e.g., concentration of amino acids, or glucose, or lactate.
20. To quantify the baculovirus using qPCR with SYBR Green, a
specific gene sequence of the bacmid DNA must be selected,
and corresponding primers need to be designed. In addition,
the copy number needs to be calculated using the following
equation:
Copy Number ¼
Amount ng
ð Þ Â 6:022 Â 10
23
Length bp
ð Þ Â 1 Â 10
9
 660
The Amount (ng) corresponds the amount of DNA.
6.022 Â 10
23 is the Avogadro’s constant, which indicates the
number of particles in 1 mol. The Length (bp) is the length of
the DNA in bp. 1 Â 10
9 represents the conversion factor for
ng, and 660 is the average mass of 1 bp double stranded DNA.
21. For performing a viral plaque assay, Sf-9 Insect Cells (e.g., Sf-9
Insect Cells Novagen, Merck, Darmstadt, Germany) are
required. For this purpose, the insect cells are cultivated semiadherently in T-bottles, as specified by the manufacturer.
22. The TCID 50 is determined, for example, by the cytopathic
effect of insect cells, or by a reporter gene that codes for a
fluorescent protein, such as the green fluorescent protein
(GFP).
23. Use a multichannel pipette for the virus dilutions 10
À0 to 10
À7
in order to process all samples simultaneously.
24. It is recommended, to attach one type of Luer lock adapter to
the tubes connected to the headplate (e.g., male Luer lock),
and the opposite adapter (e.g., female Luer lock) to all tubes
connected to bottles. This prevents confusion.
116
Julie Harnischfeger et al.