ICSE assay
Gene expression systems that use baculoviruses for gene transfection, for example BacMam system, are highly dependent on multiplicity of infection (MOI), a quantity that states how many infections viral particles there are for each cell.
MOI = ivp/cells.
While cell count can be found very easily using cell counters, for example TC10 automated cell counter in our laboratory, counting baculoviruses is much more complicated.
Several methods exist for baculovirus quantification but we use image-based cell size estimation assay which was developed in our lab and is a successor of the previously used assay. Image-based cell size estimation assay and the previous use exactly the same sample preparation techniques and analysis of cell diameter data but image-based cell size estimation assay is faster, requires less manual labor and skill and can also be used to look deeper into the infection mechanism of baculoviruses.
MOI = ivp/cells.
While cell count can be found very easily using cell counters, for example TC10 automated cell counter in our laboratory, counting baculoviruses is much more complicated.
Several methods exist for baculovirus quantification but we use image-based cell size estimation assay which was developed in our lab and is a successor of the previously used assay. Image-based cell size estimation assay and the previous use exactly the same sample preparation techniques and analysis of cell diameter data but image-based cell size estimation assay is faster, requires less manual labor and skill and can also be used to look deeper into the infection mechanism of baculoviruses.
The principle
It has been shown that baculoviruses can induce cell size growth of Sf9 cell upon infection. This growth will take around 24 h and is directly observable with a bright-field microscope. Using ICSE Tools software these bright-field images of Sf9 cells can be used to calculate the mean cellular diameter of cells on a particular image. When different serial dilutions of baculoviruses are used to infect the cells different percentages of cells get infected. In dilute points few of the cells get infected, in concentrated points almost all of the cells get infected. Therefore, the growth of average cell diameters in smaller when the baculovirus samples are dilute. When measuring the cell diameters for a whole serial dilution series, a dilution factor vs diameter graph can be plotted and fitted to calculate the baculovirus concentration.
The protocol
Prepate three-fold serial dilutions from 100.30 to 105.07 in the medium (in EX-CELL 420 serum-free medium for insect cells). Count the Sf9 cells and dilute to a concentration of 8 × 105 cells/mL, about 6.5-7 mL of the suspension is needed. Add 250 µL of the cell suspension to each well of a BioLite 24-well plate (ThermoFisher Scientific), make sure the cells are evenly distributed and maintain it at 27°C for 20–30 min. Add 250 µL of the corresponding dilution of the virus suspension to each well. Add 250 microliters of medium without virus to two of the wells to obtain mock-infected cells for comparison. Maintain the plate at 27°C until the beginning of the measurement. After 24 h place the microplate under the microscope and take 4 images of each well. If you are using Cytation5 (BioTek Instruments) then you can download the measurement protocol from here. Conduct the image analysis using ICSE Tools and generate a GraphPad Prism file with the diameter data. You can find most recent information and tutorials for ICSE Tools here. Use the sigmoidal dose response model from Analyze -> Nonlinear regression -> Dose-response – Stimulation. Alternatively you can import the same model from the Prism file here, but it will also report you the viral titre and MOI as a result of fitting.