3.6.2 Plaque Assay
The performance of a viral plaque assay using Sf-9 insect cells is
carried out according to the protocol of Bac-to-Bac™ Manual of
Life Technologies [6].
3.6.3 Determination
of the TCID 50
1. Determine the cell density of Sf-9 cells from a shake flask as
described in Subheading 3.4.2.
2. For each sample of baculovirus, dilute exponentially growing
Sf-9 cells with a fresh medium to a density of 1 Â 10
5 cells/mL
in a total volume of 10 mL.
3. Transfer 10 mL of the cell suspension into a reagent reservoir.
4. Fill 100 μL cell suspension in each well of a 96-well plate, using
a multichannel pipette (one 96-well plate is required as a seeding plate for each virus sample).
5. Incubate at 28
C for 4 h.
6. In the meantime, carry out the following:
(a) Take a second 96-well plate for the dilution of the virus
sample (per virus sample eight wells are required).
(b) Add 135 μL of fresh medium into seven of the eight
required wells (an undiluted virus sample will be placed
in the first well).
(c) Add 150 μL of the virus sample to the first well and
resuspend the mixture with the pipette.
(d) Change the pipette tip for a new one.
(e) Transfer 15 μL from the first well to the second well and
resuspend the mixture again (see Note 23).
(f) Change the pipette tip for a new one.
7. Retry the steps (c)–(f) until the eighth well is reached.
8. Create 12 replicates for each virus dilution by adding 10 μL of
the diluted virus sample from the virus dilution plate to the cell
seed plate using a multichannel pipette.
9. Incubate the cells at 28
C for 120 h.
10. Identify infected cells in each well by detecting the reporter
protein GFP.
11. Label infected cells, for example with +, and count the infected
wells per column.
12. For the calculation of the TCID 50 use the following Eq. 1:
log TCID 50
ð
Þ¼ x
j j þ D Sp À 0:5
ð
Þ
ð 1Þ
with |x| ¼ log of 100% infected wells in the last column,
D ¼ log of the dilution factor (in a total volume of 300 μL
suspension, 30 μL virus suspension results in a dilution factor
of 10, consequently log(10) ¼ 1), and Sp ¼ sum of the fraction
of the first column with 100% infected wells to the column with
0% infected wells.
Antigen Production in Insect Cells
111
The performance of a viral plaque assay using Sf-9 insect cells is
carried out according to the protocol of Bac-to-Bac™ Manual of
Life Technologies [6].
3.6.3 Determination
of the TCID 50
1. Determine the cell density of Sf-9 cells from a shake flask as
described in Subheading 3.4.2.
2. For each sample of baculovirus, dilute exponentially growing
Sf-9 cells with a fresh medium to a density of 1 Â 10
5 cells/mL
in a total volume of 10 mL.
3. Transfer 10 mL of the cell suspension into a reagent reservoir.
4. Fill 100 μL cell suspension in each well of a 96-well plate, using
a multichannel pipette (one 96-well plate is required as a seeding plate for each virus sample).
5. Incubate at 28
C for 4 h.
6. In the meantime, carry out the following:
(a) Take a second 96-well plate for the dilution of the virus
sample (per virus sample eight wells are required).
(b) Add 135 μL of fresh medium into seven of the eight
required wells (an undiluted virus sample will be placed
in the first well).
(c) Add 150 μL of the virus sample to the first well and
resuspend the mixture with the pipette.
(d) Change the pipette tip for a new one.
(e) Transfer 15 μL from the first well to the second well and
resuspend the mixture again (see Note 23).
(f) Change the pipette tip for a new one.
7. Retry the steps (c)–(f) until the eighth well is reached.
8. Create 12 replicates for each virus dilution by adding 10 μL of
the diluted virus sample from the virus dilution plate to the cell
seed plate using a multichannel pipette.
9. Incubate the cells at 28
C for 120 h.
10. Identify infected cells in each well by detecting the reporter
protein GFP.
11. Label infected cells, for example with +, and count the infected
wells per column.
12. For the calculation of the TCID 50 use the following Eq. 1:
log TCID 50
ð
Þ¼ x
j j þ D Sp À 0:5
ð
Þ
ð 1Þ
with |x| ¼ log of 100% infected wells in the last column,
D ¼ log of the dilution factor (in a total volume of 300 μL
suspension, 30 μL virus suspension results in a dilution factor
of 10, consequently log(10) ¼ 1), and Sp ¼ sum of the fraction
of the first column with 100% infected wells to the column with
0% infected wells.
Antigen Production in Insect Cells
111