Determination of baculovirus titers
The baculovirus stock titer was measured with a Baculovirus rapid titer
kit (TaKaRa, Tokyo, Japan) according to the manufacturer’s instructions.
Briefly, samples diluted from 10-3 to
10-5 were added to Sf9 monolayer cells in a 96-well
plate. After incubation at room temperature (RT) for 1 h, the
supernatant was replaced with a methyl cellulose overlay. Twenty-seven
hours later, the plate was fixed with 4% paraformaldehyde, and blocked
with normal goat serum. An Autographa californica multiple
nuclear polyhedrosis virus (AcMNPV) envelope glycoprotein (gp64) mAb
(1:200) was used as the primary antibody, and an HRP-conjugated goat
anti-mouse antibody (1:250) was used as the secondary antibody. Blue
peroxidase substrate was added and incubated for 3 h at RT. Blue-stained
foci in the wells of the highest dilution that contained a reasonable
number of foci were counted, and the titer was determined.