Editing Protein Production using Baculovirus (section)

== <br>Transfecting sf9 Insect Cells<br>  ==

<br>Use Cellfectin II, Lipofectamine or GeneExpresso for transfection. 

Grow sf9 cells in Grace’s Insect Cell Culture Medium, Unsupplemented (no antibiotics, no FBS), or Sf-900 II SFM or Sf-900TM III SFM in 6-well tissue culture plates 

Make sure cels are 95% viability and are growing in the logarithmic phase with a density of 1.5 X10<sup>6</sup> ~ 2.5X10<sup>6</sup> cells/ml 

if Sf9 or Sf21 insect cells are cultured in Supplemented Grace’s Insect Medium containing 10%FBS, use the following protocol to prepare cells for<br>transfection in a 6-well format. All amounts and volumes are given on a per well basis. 

1. Verify that the Sf9 or Sf21 cells are in the log phase (1.5–2.5 X 10<sup>6</sup> cells/ml) with greater than 95% viability. 

2. If the cell density is in range of 1.5 X 10<sup>6</sup>–2.5 X 10<sup>6</sup> cells/ml and the culture is without antibiotics, proceed to step 2a. If the cell density is not in this range or the cell culture contains antibiotics, follow steps 2b–2c: 

<br>a. Add 2 ml of Grace’s Insect Medium, Unsupplemented (without antibiotics and serum) in each well. Seed 8 × 10<sup>5</sup> Sf9 or Sf21 cells from Step 1 per well. 

Do not change medium or wash the cells. The medium carried over will enhance the transfection efficiency. Allow cells to attach for 15 minutes at<br>room temperature in the hood. Proceed to step 3. 

b. Prepare 10ml plating medium by mixing 1.5 ml Supplemented Grace’s Insect Medium containing 10%FBS (without antibiotics) and 8.5 ml Grace’s<br>Insect Medium, Unsupplemented (without FBS and antibiotics). 

c. Plate 8 × 105 Sf9 or Sf21 cells from Step 1 per well. Allow cells to attach for 15 minutes at room temperature in the hood. Remove the medium. Add<br>2.5 ml plating medium from step 2b per well. Proceed to step 3. 

3. For each transfection sample, prepare complexes as follows: 

a. Mix Cellfectin II before use, and dilute 8 μl in 100 μl Grace’s Medium, Unsupplemented (without antibiotics and serum). Vortex briefly to mix.<br>Note: You may leave this mixture at room temperature for up to 30 minutes. 

b. Dilute 1 μl baculovirus DNA in 100 μl Grace’s Medium, Unsupplemented (without antibiotics and serum). Mix gently. 

c. Combine the diluted DNA with diluted Cellfectin II (total volume ~210 μl). Mix gently and incubate for 15–30 minutes at room temperature. 

4. Add ~210 μl DNA-lipid mixture or transfection mixture (Step 3c) dropwise onto the cells from Step 2. Incubate cells at 27°C for 3–5 hours. 

5. Remove the transfection mixture and replace with 2 ml of complete growth medium (e.g., Grace’s Insect Medium, Supplemented and 10% FBS). Using<br> antibiotics is optional. 

6. Incubate cells at 27°C for 72 hours or until you see signs of viral infection. 

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{| width="822" cellspacing="1" cellpadding="1" border="1"
|-
| Signs of Infection<br> 
| Phenotype<br> 
| Description<br>
|-
| rowspan="2" | Early (first 24 hours) 
| Increased cell diameter <br> 
| A 25–50% increase in cell diameter may be seen.
|-
| Increased size of cell nuclei <br> 
| Nuclei may appear to "fill" the cells.
|-
| rowspan="3" | Late (24–72 hours) 
| Cessation of cell growth <br> 
| Cells appear to stop growing when compared to a cell-only control.
|-
| Granular appearance 
| Signs of viral budding; vesicular appearance to cells.
|-
| Detachment <br> 
| Cells release from the plate or flask.<br>
|-
| Very Late (&gt;72 hours)<br> 
| Cell lysis <br> 
| Cells appear lysed, and show signs of clearing in the monolayer.<br>
|}