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).