cassette marks transformed clones) should be picked. If this is
not possible, restriction digestion can be performed with randomly picked clones and corresponding restriction enzymes.
6. Alternatively, the titer of the generated baculovirus stock P1
can be determined after transfection. Amplification is then
performed with a specific multiplicity of infection (MOI).
7. Specific genetic elements, that can be integrated into the transfer vector, can be of great benefit. For example, a lacZ cassette
enables a blue–white screening to identify bacterial clones after
the transformation with the transfer vector. A reporter gene is
useful for carrying out a TCID 50 , as it codes for a fluorescent
protein, allowing infected cells to be identified (see Subheading
3.6.3). Another example is a secretion signal that allows for the
target protein to be secreted from the cell and that facilitates
protein harvesting.
8. Preheating the insect cell culture medium to 37
C before use
may damage cell culture medium components. The insect cell
culture medium is designed for cultivation temperatures at
27–28
C, and preheating refrigerated medium is not
necessary.
9. Add fresh medium immediately after aspiration in order to
avoid cell damage.
10. Do not seed Sf-9 cells below 0.5 Â 10
6 cells/mL in order to
avoid reduced growth or even the death of the culture.
11. Living cells appear as bright spheres with a smooth round shape
and are not stained by trypan blue. Dead cells appear as blue
spheres, as the trypan blue is able to permeate the membrane of
damaged cells. For cultivating or freezing Sf-9 cells, a viability
of >90% is appropriate.
12. Calculate the cell concentration in accordance to the manufacturer’s instructions. Consider the dilution of the cell suspension with PBS and trypan blue in the dilution factor. Usually,
the cell concentration can be calculated as follows:
Cell concentration in cells=mL ¼
Counted Cells  Chamber Factor  Dilution Factor
Number of Counted Greater Squares
13. With an initial cell concentration of 0.5–1 Â 10
6 cells/mL, cell
densities around 0.6–1 Â 10
7 cells/mL can be expected after
3–4 days of incubation.
14. After mixing the cells with preconditioned medium, add fresh
medium and the cryoprotectant DMSO. Proceed in a timely
manner, to prevent cell damage.
15. For transfection, use baculoviral DNA in a concentration of
500 ng/μL. For this, the bacmid DNA concentration must be
measured at λ ¼ 260 nm after isolation by using a plate reader
Antigen Production in Insect Cells
115
not possible, restriction digestion can be performed with randomly picked clones and corresponding restriction enzymes.
6. Alternatively, the titer of the generated baculovirus stock P1
can be determined after transfection. Amplification is then
performed with a specific multiplicity of infection (MOI).
7. Specific genetic elements, that can be integrated into the transfer vector, can be of great benefit. For example, a lacZ cassette
enables a blue–white screening to identify bacterial clones after
the transformation with the transfer vector. A reporter gene is
useful for carrying out a TCID 50 , as it codes for a fluorescent
protein, allowing infected cells to be identified (see Subheading
3.6.3). Another example is a secretion signal that allows for the
target protein to be secreted from the cell and that facilitates
protein harvesting.
8. Preheating the insect cell culture medium to 37
C before use
may damage cell culture medium components. The insect cell
culture medium is designed for cultivation temperatures at
27–28
C, and preheating refrigerated medium is not
necessary.
9. Add fresh medium immediately after aspiration in order to
avoid cell damage.
10. Do not seed Sf-9 cells below 0.5 Â 10
6 cells/mL in order to
avoid reduced growth or even the death of the culture.
11. Living cells appear as bright spheres with a smooth round shape
and are not stained by trypan blue. Dead cells appear as blue
spheres, as the trypan blue is able to permeate the membrane of
damaged cells. For cultivating or freezing Sf-9 cells, a viability
of >90% is appropriate.
12. Calculate the cell concentration in accordance to the manufacturer’s instructions. Consider the dilution of the cell suspension with PBS and trypan blue in the dilution factor. Usually,
the cell concentration can be calculated as follows:
Cell concentration in cells=mL ¼
Counted Cells  Chamber Factor  Dilution Factor
Number of Counted Greater Squares
13. With an initial cell concentration of 0.5–1 Â 10
6 cells/mL, cell
densities around 0.6–1 Â 10
7 cells/mL can be expected after
3–4 days of incubation.
14. After mixing the cells with preconditioned medium, add fresh
medium and the cryoprotectant DMSO. Proceed in a timely
manner, to prevent cell damage.
15. For transfection, use baculoviral DNA in a concentration of
500 ng/μL. For this, the bacmid DNA concentration must be
measured at λ ¼ 260 nm after isolation by using a plate reader
Antigen Production in Insect Cells
115