Incubate co-transfected monolayers for 12-24 h at 28oC. Then add 1 ml of medium supplemented with antibiotics, for example, TC-100 medium (Sigma, Cat# T3160) supplemented with heat inactivated 10% Fetal Bovine Serum and 1% penicillin/streptomycin solution (Invitrogen SKU# 15070-063) and continue incubation at 28oC. Synthetic serum free media can also be used, for example, Insect-xpress (Lonza) or SF-900 (Invitrogen), contingent upon the medium used to overlay monolayers for the transfection. To prevent evaporation during the incubation, placing the monolayers into a humid container (any plastic container containing wet filter paper) is recommended. Alternatively, use a humidified incubator.
Harvest recombinant baculovirus stock after a total incubation time of 60-72 h post co-transfection. At this time, the virus titer will be typically 106-107 per ml, a P0 stock. AB Vector recommends amplification to a high titer P1 stock prior to expression tests to ensure sufficient virus to provide 100% infection of cell monolayers.
|Technology - Maintaining cells - Transfection - Baculovirus stocks - Protein expression|