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 10⁶ 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 ml 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 ml 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.