Smm 293 tii medium

Codon-optimized cDNA for full-length human Na_v1.7 (Uniprot Q15858), a gift from Tsinghua University, was cloned into the pCAG vector with Twin-Strep-tag and Flag-tag in tandem at the amino terminus while codon-optimized cDNAs for β1 subunit (Uniprot Q07699) and β2 subunit (Uniprot O60939) were cloned separately into the pCAG vector without affinity tag. All the plasmids for transient expression were verified by DNA sequencing. HEK293F suspension cells (Thermo Fisher Scientific, R79007) were cultured in SMM 293T-II medium (Sino Biological Inc.) at 37 °C, supplied with 5% CO₂ under 60% humidity, and transfected with plasmids when the cell density reached 1.5–2.0 × 10⁶ cells per ml. For one-liter cell culture, a mixture of expression plasmids including 1.5 mg plasmids for Na_v1.7, 0.5 mg plasmids for β1, and 0.5 mg plasmids for β2 was pre-incubated with 4 mg 40-kDa linear polyethylenimines (Polysciences) in 50 ml fresh medium for 15–30 minutes, and then added to the cell culture for transient expression of human Na_v1.7 complex.

CHO/dhFr⁻ cells (12200036, Cell Bank of the Chinese Academy of Sciences, Shanghai, China) and the cell lines derived from them were propagated in IMDM medium (HyClone) containing 10% fetal bovine serum (HyClone), 0.1 mM hypoxanthin, and 0.016 mM thymidine (HT, Gibco, USA), at 37 °C in a 5% CO₂ incubator. The suspension Expi293F cells (Invitrogen, Carlsbad, CA, USA) were cultured in SMM 293-TII medium (Sino BiologicalInc, Beijing, China) in suspension at 37 °C, 5% CO₂. Cell density was maintained between 3 × 10⁵ and 3 × 10⁶ cells/ml by dilution of the cell suspension in the same growth medium.

Viruses used in this study comprised wild-type isolates and reassortants, containing internal genes from A/Puerto Rico/8/1934, which were developed as candidate vaccine viruses for vaccine manufacturing (Fig. 1a). Viruses were propagated in Madin–Darby canine kidney (MDCK) cells or in embryonated eggs. For passive protection studies in mice, wild-type A/Shenzhen/TH002/2016 (H5N6) was amplified in embryonated eggs^{43 (link)}. Virus titers were determined by end point dilution in MDCK cells (TCID₅₀). MDCK cells were obtained from the American Type Culture Collection (Manassas, VA, USA) and cultured in Dulbecco’s modified Eagle’s medium (DMEM; Gibco, cat. no. 11965) with or without 10% fetal bovine serum (FBS). HEK293F cells were grown in suspension in SMM 293-TII medium (Sino Biological, Cat# M293TII) at 37 °C in a humidified 5% CO₂ incubator rotating at 130 rpm. Sf9 and Hi5 insect cell line from Invitrogen were cultured at 27 °C. All cell lines were tested negative for mycoplasma contamination.

HCC cell lines (Hep3B, HCCLM3, Huh7 and MHCC-97L) were purchased from the cell bank of the Chinese Science Academy. HepG2 and HEK293T cells were obtained from the American Type Culture Collection. DR-GFP and EJ5 U2OS cells were a gift from Dr. Sijie Liu (Peking University, China). All these cells were cultured in DMEM (WISENT, China), containing 10% FBS with 1% penicillin streptomycin. HEK293F cells were cultured in SMM 293-TII medium (Sino Biological, China) in a shaker incubator at 120 rpm. All cells above were maintained at 37 °C and 5% CO2 in a humidified incubator.

HEK293F cells (Invitrogen) were cultured in SMM 293T-II medium (Sino Biological Inc.) at 37 °C under 5% CO₂ with agitation at 120 rpm. When the cell density reached 1 × 10⁶ cells per mL, the plasmid containing IS607 TnpB and reRNA were transfected into the cells. For 10 mL of the transfection system, 10 μg of plasmid was pre-mixed with 100 μg of 25-kDa linear polyethylenimines (Polysciences) in 1 mL fresh medium for 30 min before transfection, followed by dilution of the cell culture to 0.7 × 10⁶ cells per mL with fresh medium. Transfected cells were cultured at 37 °C under 5% CO₂ with agitation at 120 rpm for 72 h before they were collected.