Sf9 cells

Sf9 cells (Gibco) were grown in suspension culture at 27°C in 500 mL of SFM900III medium (Invitrogen) in 1L Corning Erlenmeyer Flasks (Ref: 431147) under rotation speed of 170 rpm on INFORS HT Celltron shaker. Baculoviruses were generated from recombinant bacmids according to the guidelines of the Bac-to-Bac protocol and were amplified in 150 mL of suspension cultures of Sf9 cells at a density of 10⁶ cells per mL in 250 mL Corning Erlenmeyer Flasks (Ref: 431144). rAAV productions were performed by dual infection of baculoviruses harboring the recombinant AAV genome (γSGC or mSeAP) and the AAV rep/cap genes, each at a MOI of 0.05 (PFU titer) in 150 mL of Sf9 cell culture seeded at 10⁶cells.mL^-1 in 250 mL Erlenmeyer Flasks. At 96 h p.i. 1 mL of the total culture was recovered for direct quantification of rAAV production prior to purification. Concerning rAAV productions performed using the WT baculovirus, E64 protease inhibitor (Sigma #E3132) was added to a subset of cell cultures to a final concentration of 50 μM at different times post-infection (0h, 24h, 48h, 72h, 96h, and post cell lysis for rAAV recovery). wtAAV2 has been produced as described by Zeltner et al. [18 (link)], with the pDG2 plasmid [19 (link)] and the WT AAV2 sequence (NC_001401.2) obtained by gene synthesis from the Genewiz company and cloned into the pGRG25 plasmid [20 (link)].

Sf9 cells (Procell, Wuhan, China) were cultured in insect-cell medium (Sino Biological, Beijing, China) in a cell-culture flask at 27 °C. When the cell confluence reached 80–90%, cells were resuspended in insect-cell medium, and the cell density was adjusted to 2 × 10⁵ cells/ml. Sf9 cells (100 μl) were added to each well of a 96-well plate (Corning, Guangzhou, China) and incubated at 27 °C for 24 h. Different final concentrations of rHtA (0, 1, 2, 4, 8, 16, 32, and 64 ng/ml) were added and incubated at 27 °C for 48 h, and cell proliferation was determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Briefly, 10 μl MTT solution (5 mg/ml MTT in PBS) was added to each well of the 96-well plate, followed by incubation at 27 °C for 4 h, after which media supernatants were discarded carefully. Dimethyl sulfoxide (100 μl) was then added to each well to dissolve the formazan for 10 min at 37 °C. Absorbance was measured on an ELISA plate reader (BioTek ELX800, BioTek Instruments Inc., Vermont, USA) at a wavelength of 570 nm.