The DIY Baculovirus Expression System

Contents

• Introduction
• Subculturing SF9 Cells
• Infecting insect cells for protein expression

Introduction to the DIY Baculovirus Expression System

The BV group will maintain an SF9 inoculum using the incubators in 5S26 for use in DIY BV expression. The upper incubator is for Laurence’s group, the lower incubator is for Davids group. On Friday, take 3 roller bottles from the appropiate incubator to 5C8, pool the cells and count. Subculture the Sf9 cells using an MSC and then return the roller bottles to your incubator in 5S26 to grow over the weekend. On Monday, take your expanded Sf9 culture to 5C8 and infect with virus. Return the infected Sf9 cells to 5S26 harvested after the appropriate length of incubation. Please check there is sufficient virus in stock for the infection you wish to carry out before making a DIY booking. Let the BV group know if viral stocks are running low after your infection.

Subculturing SF9 Cells

Please work aseptically throughout. This includes wearing the red collared tissue culture coats provided in 5C8. Spray all surfaces of the safety cabinets with 70% ethanol. Hold the tops of media and roller bottles face down. Take care not to touch the necks of the roller bottles.

Counting Sf9 cells

1. In the safety cabinet,combine and mix the following:
   0.5mls 0.4% Trypan blue + 0.3mls 1xPBS + 0.2mls SF9 cells.
2. Dampen the haemocytometer (this can be done by breathing on it) and press the coverslip firmly into place. Check for Newtons rings.
3. Mix the cell suspension again. Use a P20 to take up 10µl and add this to one chamber. Repeat for the second chamber.
4. Count all the cells in the large central square which is 5 squares across divided into 16 squares. Count a further 3 large squares (also divided into 16 squares). Repeat for second chamber.

NB: Don't include in your count any cells which are blue - they are non viable.

Average number of cells/ml=Average number of cells per large square x 5x10⁴

Example

Counts of 35,36,39,40 (chamber 1) 30,32,37,38 (Chamber 2)

Total count = 287

Average count = 278/8 = 36

Average number of cells/ml = 36 x 5x10⁴ = 1.8x10⁶ cells/ml

Splitting the cells

Sf9 cells double approximately every 18-24 hours. A cell density of 2x10⁶ cells/ml is preferable for protein expression in Sf9 cells.
Split the cells to a density of 2.5x10⁵ cells/ml. Over 3 days this should give a density of 2x10⁶ cells/ml.

1. Add 400mls of insect cell media SF900II to each roller bottle, plus 1ml of Pen/Strep. Add the desired volume of cell innoculum to give a density of 2.5x10⁵ cells/ml.
   
   Volume to add = Volume of media * Desired final cell count/ (Inoculum cell count - desired final cell count)
   
   Example: Inoculum cell count is 1.7x10⁶ cells/ml. We must add 69ml of inoculum to 400ml of media to reach the desired cell count.
   
   Volume = 400 * 2.5x10⁵ / (1.7x10⁶ - 2.5x10⁵) = 69ml.
   
2. Incubate the roller bottles at 29°C for 3 days.

Infecting Insect Cells for Protein Expression

Sf9 cells are generally infected with an MOI (multiplicity of infection) of 2. This seems to be optimum for protein expression.

1. Count the cells after the 3 day incubation and calculate how much virus to add to each bottle of cells.
   
   Volume required = MOI x culture cell count x culture volume / viral titer.
   
   Example: Cell density after 3 days is 2x10⁶ cells/ml
   Volume of culture is 469ml
   Viral titer is 5x10⁸ Pfu/ml
   Volume of virus required = 2 x 2x10⁶ x 469 / 5x10⁸ = 3.8ml
   
   
2. Incubate and harvest cells after 3 days.