Protocol 8: SDS-PAGE and western blot analysis of GFP expression
3 Results
3.2 Analysis of infection efficiency and titer of the amplified baculovirus using
After infection for 120 hours, the collected amplified baculovirus stocks were separated from detached cells by a using centrifuge at the centrifugal force of 500 x g for 5 minutes. Amplified virus stocks generated using 20 μl and 40 μl of P3 recombinant virus were termed as P4V1 and P4V2, respectively.
3.2.1 Fluorescence microscopic analysis of Sf9 cells infected with the amplified baculovirus stocks
To investigate the infection efficiency of the two amplified virus stocks, Sf9 cells were seeded into 24-well plates on glass coverslips for 24 hours and infected with 10 μl, 25 μl and 50 μl of P4V1 and P4V2 each (see fig. 3.1A). 48 hours post-infection, the cells were fixed using paraformaldehyde (PFA) and analysed under the fluorescence microscope. The number of cells infected with recombinant baculovirus containing the gene of GFP was followed by its green fluorescence in order to determine the infection efficiency. During the fluorescence microscope analysis, nuclei of the Sf9 cells grown on the coverslip were observed with the DAPI filter (DNA stain) while infected cells were observed using the FITC filter (GFP). Then, images of the same area were captured using both filters and the overlay of both images was created using Adobe Photoshop software.
Images of cells grown on glass coverslips and infected with different amounts (10 μl, 25 μl and 50 μl) of P4V1 and P4V2 are shown in figure 3.4 and 3.5, respectively. It can be observed in both figures that higher amounts of virus stocks used for infection produce higher number of green fluorescence signals from expressed GFP protein for both virus stocks. Next, the infection efficiencies of both virus stocks were calculated to determine the optimal virus stock with the highest virus concentration. To calculate the infection efficiency of P4V1 and P4V2, the total number of cells in the DAPI filtered image (blue) and infected cells of the same area of the coverslip from the FITC filtered image (green) were counted. However, in this work, only 25 % of the area (as shown in Figure 3.4 and 3.5 with red-colored square) of each image was considered for the infection efficiency calculations.
After counting the number of total and infected cells from each image, the infection efficiencies of 10 μl, 25 μl and 50 μl of P4V1 and P4V2 virus stocks are shown in figure 3.6. It is clear from this calculation that for virus stock P4V1, when 10 μl of the virus was used for infection, 5.0 % of the cell population was infected. When the volume of P4V1 virus was increased by 2.5 and 5 times, 10.1 % and 17.4 % of the cell population was infected, respectively. Therefore, the increase of the P4V1 dose increases the infection efficiency but not in the same proportion as that of the virus dose i.e., 2.5 and 5 times of virus volume increase produced only 2 and 3.5 times of increase in infection efficiency. Similarly, infection efficiency of P4V2 for volumes of 10 μl, 25 μl and 50 μl was 11.8
%, 23.5 % and 47.9 %, respectively. That means, when the dose of P4v2 was increased by 2.5 and 5 times, the infection efficiency increased by 2.2 and 4.5 times, respectively. So, there is a closer
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DAPI FITC OVERLAY
10 μl
25 μl
50 μl
Figure 3.4: Fluorescence microscopy analysis of Sf9 cells, grown on glass coverslips and infected with different amounts of amplified baculovirus stock P4V1. Images (A), (D) and (G) represent DAPI stained cell nuclei (blue) of the coverslip to which 10 μl, 25 μl and 50 μl volumes of P4V1 were added, respectively.
These images were captured using the DAPI filter at 90 ms exposure time and 100 x magnification. Images B, E and H show the fluorescence signals from the expressed GFP when cells were infected with 10 μl, 25 μl and 50 μl of P4V1, respectively. They were captured using the FITC filter at 1400 ms exposure time and 100 x magnification. Images (C), (F) and (I) are overlays made by Adobe Photoshop software of corresponding filtered images of (A-B), (D-E) and (G-H), respectively. For the calculation of infection efficiency, only 25 % of the region [as shown in a red square as an example in image (C)] of each image was considered.
A B C
D E F
G H I
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DAPI FITC OVERLAY
10 μl
25 μl
50 μl
Figure 3.5: Fluorescence microscopy analysis of Sf9 cells, grown on glass coverslips and infected with different amounts of amplified baculovirus stock P4V2. Images (A), (D) and (G) represent DAPI stained cell nuclei (blue) of the coverslip to which 10 μl, 25 μl and 50 μl volumes of P4V2 were added, respectively.
These images were captured using the DAPI filter at 90 ms exposure time and 100 x magnification. Images B, E and H show the fluorescence signals from the expressed GFP when cells were infected with 10 μl, 25 μl and 50 μl of P4V2, respectively. They were captured using the FITC filter at 1400 ms exposure time and 100 x magnification. Images (C), (F) and (I) are overlays made by Adobe Photoshop software of corresponding filtered images of (A-B), (D-E) and (G-H), respectively. For the calculation of infection efficiency, only 25 % of the region [as shown in a red square as an example in image (C)] of each image was considered.
A B C
D E F
G H I
38 correlation between the increase in dose and infection efficiency of P4V2 as compared to P4V1. Furthermore, it can be concluded that virus stock P4v2 is more potent than virus stock P4v1 when infection efficiencies of the same infection virus volumes are compared. The highly efficient virus stock P4V2 gives also stronger protein expression signals. For this reason, P4v2 virus stock was chosen for the later infection in large scale culture (in this work spinner flasks) of Sf9 cells for the determination of protein expression. This infection efficiency data is used later to estimate the titer of the virus stock in the following section.
Figure 3.6: Infection efficiencies of the amplified baculovirus stocks P4V1 (left) and P4V2 (right) as determined by fluorescence microscopic analysis. Before fluorescence analysis cells were infected with 10, 25, 50 μl virus stock for 48 hours on glass coverslips. Only 25 % area of the images taken from the fluorescence microscope were considered for the calculation of infection efficiencies. It can be observed that an increment in the dose of virus leads to an increase in the infection efficiency for both the virus stocks.
3.2.2 Estimation of baculovirus titer and Multiplicity of Infection (M.O.I)
After analysing the infection efficiency of virus stock P4V2, it is required to calculate the virus titer. First step of this process is to calculate the total number of cells on the glass coverslip which is determined by the following steps:
i. Total Surface area of the coverslip (A):
Diameter of the cover slip (d) =11000 μm
Therefore, the total surface area of the cover slip (A) = (3.14 / 4) x (d)2
= (3.14 / 4) x (11000)2
= 94,985,000 μm2
ii. Total surface area of the image captured by the fluorescence microscope (A2):
Height (h) and width (w) of the image from the fluorescence microscope obtained are 850 μm and 650 μm, respectively.
5.0%
10.1%
17.4%
0%
5%
10%
15%
20%
10 μl 25 μl 50 μl
Infection efficiency
Volume of virus stock P4V1
Infection efficiency after infection with increasing volumes of virus stock P4V1
11.8%
23.5%
47.9%
0%
10%
20%
30%
40%
50%
60%
10 μl 25 μl 50 μl
Infection efficiency
Volume of virus stock P4V2
Infection efficiency after infection with increasing volumes of virus stock P4V2
39 (A2) = 850 x 650 μm2
= 552,500 μm2
iii. Surface area of the area under consideration (A3) was 25 percent of the total surface area of the image
(A3) = 425 x 325 μm2 = 138,125 μm2
iv. Number of counted cells on a coverslip, infected with lowest amount (10 μl) of P4V2 virus stock for the area under consideration (b) = 471 cells
Hence, total number of cells on the coverslip = (b x A) / (A3) = 323,894 cells.
Based on the infection efficiency calculation as mentioned in (figure 3.6) for the virus stock P4V2,
it was shown that 11.8 % of total cells infected with 10 μl of virus stock. Therefore, virus titer estimation calculation of virus stock P4V2 is as followed.
𝑇𝑖𝑡𝑒𝑟 𝑜𝑓 P4V2 virus stock = (Infection rate x total cells) amount of virus stock used 𝑇𝑖𝑡𝑒𝑟 𝑜𝑓 P4V2 virus stock = (11.8 % x 323,894 cells)
10 µl
= 3821 infected cells / μl 𝑇𝑖𝑡𝑒𝑟 𝑜𝑓 P4V2 virus stock ≈ 3.8 x 106 PFU/ ml
For spinner flaks with 50 ml of Sf9 cell culture, the total number of cells was 5.0 x 107. Three different spinner flasks, contained 50 ml of Sf9 cell suspensions, were infected with 1ml, 5 ml and 10 ml of P4V2 baculovirus stock.
𝑀. 𝑂. 𝐼 = (Number of virus particles) total number of cells
Based on this equation, M.O.I of spinner flasks infected with 1 ml, 5 ml and 10 ml were 0.08, 0.4 and 0.8, respectively.
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