Sf 900 3 sfm

Sf9 cells were used to generate baculovirus stocks using the Bac-to-Bac system (Life Technologies), according to the manufacturer's instructions. A baculovirus stock carrying the gene for DNMT1 was used to infect 4–12 L of Sf9 cells at a density of 2 million cells/mL in Sf-900 III SFM (Invitrogen cat. # 12658–027). Multiple 1 L cell cultures in 4 L flasks (VWR cat. # 32645–044) were incubated for 72 h at 27°C, 130 revolutions per minute (rpm). Cells were harvested by centrifugation for 20 min at 2000 relative centrifugal force (RCF) (JLA-8.1, Beckman) at 4°C, then frozen in liquid nitrogen and stored at −80°C until protein purification.

Expression of human PRC2 was carried out as previously described (23 (link)), with some modifications. Sf9 cells where used to generate baculovirus stocks using the Bac-to-Bac system (Life Technologies), according to the manufacturer's instructions. Appropriate ratios of baculovirus stocks carrying genes for the four (with the exclusion of AEBP2) or five PRC2 subunits were used to co-infect 4–12 l of Sf9 cells at a density of 2 million cells/ml in Sf-900™ III SFM (Invitrogen cat # 12658–027). Multiple 1 l cell cultures in 4 l flasks (VWR cat # 32645–044) were incubated 72 h at 27°C, 130 revolutions per minute (rpm). Cells were harvested by centrifugation for 20 min at 2000 relative centrifugal force (RCF) (JLA-8.1, Beckman) at 4°C, frozen in liquid nitrogen and stored at −80°C until protein purification.

Marc-145 cells (African green monkey kidney cells) and HEK293T cells were cultured in Dulbecco’s minimal essential media (DMEM, Hyclone, Thermo Scientific, MA, USA) with 10% fetal bovine serum (FBS, Invitrogen, USA) at 37 °C in 5% CO₂. Porcine alveolar macrophages (PAMs) were prepared as described previously [31 (link)]. PRRSV strains including ZJfh17 (lineage 8), HNxx16 (lineage 1), ZJnb16–2 (lineage 8, 3), ZJhz16–2 (lineage 5), and JS18–3 (lineage 8) were previously isolated in CATG lab, Zhejiang University, China and titrated in PAMs. Commercially modified-live PRRS vaccines with corresponding virus strains named HuN4-F112 (Harbin Weike Biotechnology Development Company, Harbin, China) were propagated and titrated in Marc-145 cells. Sf9 insect cells were used to propagate recombinant baculoviruses in SF900 III SFM (Invitrogen, Carlsbad, CA, USA) at 27.5 °C.

The precultured Sf9 cells were diluted by serum free medium to 1×10⁶ cells /mL and then transfected by recombinant bacmid using Cellfectin II reagent (Invitrogen, USA). The recombinant bacmid was collected after 72h according to the manufacturer instruction. To produce M2e-CtxB fusion protein, the Sf9 cells were seeded at the density of 1−2 × 10⁷ per flask (75 cm²) and infected by recombinant bacmid collected from the last stage at MOI (Multiplicity of Infection) 3 in 10 mL of Sf-900 III SFM (Invitrogen, USA). All the flasks were incubated at 27 °C.