• Protocol A: Calcium phosphate co-transfection of insect cells
• Protocol B: Analysis of nonsecreted protein expression
• Protocol C: Growing and freezing of insect cells

Protocol A: Cotransfection Protocol:

1. Seed 2 x 10⁶ Sf9 or Sf21 cells onto 60 mm tissue culture plates in 4 ml and allow them to attach for 30 minutes at 27°C.
2. Aspirate the old medium from the plate and replace with 750 µl supplemented Grace's medium.
3. To a microcentrifuge tube add 0.5 µg baculoviral DNA to 2-5 µg of recombinant transfer vector. Add Calcium Phosphate Transfection Buffer to a final volume of 750 µl and mix gently.
4. While rocking the plate, add the calcium phosphate solution dropwise. You should notice the formation of a white precipitate. Incubate at 27°C four hours.
5. Aspirate the transfection mixture from the plate. Replace with 4 mls TNM-FH and incubate four to five days. Observe after three days. Infected cells will appear large and round. Harvest the supernatant and centrifuge the cellular debris at 1000xg for five minutes. The supernatant from baculovirus-infected insect cells can occasionally produce a viral stock of 10⁹ pfu/ml or greater.

Protocol B: Analysis of nonsecreted protein expression:

1. Seed 1 x 10⁶ log phase Sf9 cells in 35 mm tissue culture dishes to form an even monolayer. Incubate at 27°C for 30 minutes to ensure attachment. If performing a time course prepare one plate per time point and an additional one for a control.
2. Aspirate media and replace test plates with an appropriate amount of fresh media containing 2 x 10⁷ pfu (an MOI of 20) recombinant baculovirus. Replace the control with fresh media. Incubate for one hour at 27°C.
3. Aspirate media and replace with fresh.
4. Incubate at 27°C. At each time point aspirate media from the appropriate plate and lyse the cells using 50 µl of lysis buffer per 10⁶ cells. Add protease inhibitors to a concentration of 1X. The samples are ready for analysis by SDS-PAGE.

Protocol C: Growing and Freezing Insect Cells

Freezing Protocol

1. Pellet insect cells by gentle centrifugation at 1000xg for five minutes. Discard the old medium and replace with fresh medium to a concentration of 1-2 x 10⁷ cells/ml.
2. Mix an equal volume of the freezing medium with the cell suspension and aliquot 1.0 ml into sterile cryovials.
3. Freeze cells slowly at minus 20°C for one hour, then at minus 70°C overnight.
4. Transfer the vials to liquid nitrogen.
5. Thaw one vial to examine viability and contamination after one week.

Thawing Protocol

1. Remove the vial from liquid nitrogen and thaw rapidly by swirling in a water bath at 37°C.
2. Disinfect the vial by spraying with 70% ethanol. Transfer the vial to a laminar flow hood.
3. Aseptically transfer the contents to a 60 mm tissue culture plate containing 4 mls of complete TNM-FH insect cell medium.
4. Incubate at 27°C for three hours and observe to ensure attachment.
5. Once the cells have attached, aspirate medium and replace with 4 mls of fresh medium. Incubate at 27°C until confluent then split cells 1:3. Cells may be detached by gently blowing the monolayer with a stream of medium using a 10 ml pipette.

References

• Kitts, P. A. and R. P. Possee, (1993). BioTechniques 14(5):810-817.
• O'Reilly, D.R., L.K. Miller, and V.A. Luckow, (1994). Baculovirus Expression Vectors.