Editing Protein Production using Baculovirus (section)

== <br>Amplification P1 Baculoviral to get P2 Stock<br>  ==

Amplify P1 viral stock in a 10 ml suspension culture at 2 X10<sup>6</sup> cells/ml or in 6-well tissue culture plates at 2 X 10<sup>6 </sup>cells/well. Calculate the number of Sf9 or Sf21 cells that you will need for infection and expand cells accordingly. Make sure that the cells are healthy, of low passage (5–20), and have &gt;95% viability before proceeding to infection. 

To amplifyviral stock, infect cells at a multiplicity of infection (MOI) ranging from 0.05 to 0.1. MOI is defined as the number of virus particles per cell. Use the following formula to calculate how much viral stock to add to obtain a specific MOI: 

Inoculum required (ml) = (MOI (pfu/cell) x number of cells / titer of viral stock (pfu/ml)) 

Note: If the titer of P1 viral stock has not been determined , it is assumed that the titer ranges from 1 X 10<sup>6</sup> to 1 X 10<sup>7</sup> pfu/ml. 

To infect a 10 ml culture at 2 X 10<sup>6</sup> cells/ml using an MOI = 0.1. Assuming that the titer of P1 viral stock is 5 X 10<sup>6</sup> pfu/ml. 

Inoculum required (ml) = (0.1 pfu/cell 2 x 10<sup>7</sup> cells /5 x 10<sup>6</sup> pfu/ml)<br>Inoculum required (ml) = 0.4 ml 

Several key points for successful amplification of baculovirus: 

Use sterile P1 baculoviral stock that is free of contaminants<br>Use a low MOI between 0.05–0.1 as higher MOI will reduce baculovirus quality<br>Harvest the virus when 70–80% of cells are dead 

=== <br>Amplify P1 viral stock in a 6-well plate.<br>  ===

<br>1. On the day of infection, prepare your Sf9 or Sf21 cell suspension and plate cells at 2 X 10<sup>6</sup> cells/well. Incubate cells at room temperature for 1 hour to allow attachment. 

2. After 1 hour, inspect cells under an inverted microscope to verify attachment. 

3. Add the appropriate amount of P1 viral stock to each well. 

4. Incubate the cells for 48 hours in a 27°C humidified incubator. 

5. 48 hours post-infection, collect 2 ml of medium containing virus from each well and transfer to sterile 15 ml snap-cap tubes. Centrifuge the tubes at 500 X g for 5 minutes to remove cells and large debris and to obtain clarified baculoviral stock. 

Note: It is possible to harvest virus at later times after infection (e.g., 72 hours). Optimal harvest times can vary and should be determined for each baculoviral construct. Remember that culture viability will decrease over time as cells lyse. 

6. Transfer the supernatant to fresh 15 ml snap-cap tubes. This is the '''P2''' viral stock. Store at 4 °C, protected from light. For long-term storage, store an aliquot of the P2 stock at –80 °C, protected from light. 

7. Proceed to the next section to determine the titer of P2 viral stock. 

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