Grace s insect medium

Rescue of the recombinant baculovirus, rpFB1-Gn head-PA, was done by transfection of Sf9 cells with the recombinant bacmid, pFBac 1-HBM-Gn head-PA, using the Cellfectin II Reagent (Life Technologies, United States). Briefly, Sf9 cells were seeded 24 h prior to transfection in a six-well plate at a density of 8 × 10⁵ cells/well to achieve a confluency of 80–90%. For transfection, 3 μg of recombinant bacmid was diluted in 100 μL Grace‘s Insect Medium (Thermo Fisher Scientific, United States) and combined with 8 μL Cellfectin II Reagent diluted in Grace‘s Insect Medium to a final volume of 210 μL. The resulting transfection mixture was vortexed briefly and incubated at room temperature for 15–30 min, after which the DNA–lipid mixture was added onto the Sf9 cells and incubated at 27°C. Supernatants containing first generation (P1) recombinant baculovirus were harvested 72 h post-transfection and passaged for three additional generations on Sf9 cells to yield fourth generation (P4) recombinant baculovirus. All passaging was done upon observation of cytopathic effects (CPE), typically within 24 h post-infection. Expression of the RVFV Gn head-PA fusion gene was confirmed by PCR (primers in Table 1) for both P3 and P4 recombinant baculovirus.

Third instar larval eye-imaginal discs were dissected in Grace’s Insect Medium (ThermoFisher) then transferred into Grace’s Insect Medium containing 0.25 mg/ml BrdU (Invitrogen B23151) and incubated at 25 °C for 90 min. Discs were then washed in Grace’s Insect Medium for 5 min on ice followed by washing two times for 5 min each in 1× PBS on ice. Discs were fixed overnight (wrapped in foil) in 1% paraformaldehyde/0.05% Tween20. The following day discs were washed three times for 5 min each in 1× PBS and permeabilized for 20 min at RT in 0.3% PBST. To remove detergent, discs were washed five times for 5 min each in 1× PBS and DNAse treated for 30 min at 37 °C. Discs were then washed three times for 10 min each in 0.1% PBST and incubated overnight at 4 °C in mouse anti-BrdU primary antibody (B44) (BD, 1:50). The next day, discs were washed five times for a total of 30 min with 0.1% PBST and incubated overnight in goat anti-mouse F(ab)’2 AlexaFluor-555 secondary antibody (Cell Signaling, 1:500). Finally, discs were washed three times for 10 min each in 0.1% PBST and mounted in VectaShield anti-fade mounting medium.

Sf9 cells (ATCC^®® CRL-1711™, ATCC, Manassas, VA, USA) were grown to 80% confluence at 27 °C in Sf-900 III medium (Life Technologies, Carlsbad, CA, USA) supplemented with fetal bovine serum (FBS; 4%). The bacmid DNA containing the chimeric Nc-aD coding sequence was transfected into the Sf9 cells using the cationic lipid transfection method [43 (link)]. Briefly, in a 6-well plate, 8 × 10⁵ Sf9 cells were seeded per well and allowed to attach for 30 min. The Sf-900 III medium was aspirated, and replaced with Grace’s insect medium (2 mL; Life Technologies, Carlsbad, CA, USA). A transfection mixture consisting of Grace’s insect medium (200 µL), recombinant bacmid DNA (1 µg) and Lipofectamin 2000 (10 µL; Invitrogen, Carlsbad, CA, USA) was added to the Sf9 cells in a dropwise manner and incubated at 27 °C for 5 h. The medium was then replaced with fresh Sf-900 III medium (2 mL) supplemented with FBS (4%). The Sf9 cells were incubated at 27 °C for 4 days. The supernatant containing the baculovirus was collected and kept as stocks.