Recombinant baculovirus isolation by plaque assay
Isolation of recombinant baculovirus by viral plaque assay was performed by seeding 6-wells plate with 1ml of cell suspension at concentration equal to 1x106 viable Sf9 cells per ml. After 30 min at 27°C, attached cells were infected with serial dilutions of baculovirus. An initial dilution was realized from 50µl of the baculovirus stock in 500µl of Sf-900 III serum-free medium and sequential 1:3 dilutions were done in 48-wells plate adding 250µl of the initial dilution in 500µl of medium. Typically, to be able to quantify the lysis plaque for a baculovirus stock of >1x107 plaque-forming units (pfu) per ml, 200µl of the dilutions 7.2x10-4, 2.2x10-5 and 6.5x10-5 were added directly into each well, in duplicate. The plate was incubated for 3h at 27°C in a humidified chamber. To prepare plaquing overlay, 4ml of 4% agarose gel (ThermoFisher Scientific, MA) that was preheated at 70°C was mixed with 12ml de SF900 medium 1.3X (ThermoFisher Scientific, MA), the inoculum was removed from each well from high to low dilution and replace with 2ml of the diluted agarose. After gel solidification, the plate was carefully placed in a humidified chamber and incubated between 9 to 11 days at 27°C. Isolated clones are picked-up from the solid medium and individually amplified and finally characterized (P1).