|Preparation of monolayers for transfections|
Monolayers of Sf9 or Sf21 cells at approximately 50-60% confluency (semi-confluent monolayers) are used for transfections. Higher cell densities are not recommended as they can rapidly form overgrown confluent monolayers. Overgrown monolayers with small crowded cells are incapable of supporting efficient baculovirus replication, and a monolayer that grows to confluency early in the transfection experiment will have the recombinant virus yield dramatically reduced. Lower cell densities are also not recommended, as the virus yield will be proportionally less. Depending on the medium, cell replication and virus yield could be impeded in lower density monolayers (below 50% confluency) grown in Petri dishes and plates.
Seed monolayers of Sf9 or Sf21 cells to about 50-60% confluency in 8.8 cm2 Nunclone Delta treated Petri dishes (Nunc Cat. #150318) or in 6-well plates. We routinely seed cells from log phase suspension cultures, but cells resuspended from monolayers can also be used. Seed cells 1-2 h prior to transfection to ensure optimal cell density. Alternatively, cells could be seeded overnight prior to the transfection. However, in this case allow for a doubling of cell number during the night.
Cells grown in any media that is recommended for cultivating Sf9 or Sf21 cells can be used for transfection. However, sera and antibiotics are not compatible with transfection. Therefore, after seeding the monolayers remove the cell culture medium and wash the monolayers twice with 1 ml of sterile PBS. Do not operate more than 20 monolayers at once because of the risk of drying. Add 1 ml of the medium to the monolayer. The monolayer is now ready and can be used for transfection any time during the same day.
|Technology - Maintaining cells - Transfection - Baculovirus stocks - Protein expression|
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