Hek293f suspension cells

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.

Codon-optimized cDNAs of CACNA1E for full-length Ca_v2.3 α1 (2,313 residues, Uniprot Q15878-1), CACNA2D1 for α2δ−1 (1,103 residues, Uniprot P54289-1) and CACNB3 for β3 (484 residues, Uniprot P54284-1) were synthesized (BGI Geneland Scientific). For CH2-deleted Ca_v2.3 (ΔCH2), residues 773-791 of the α1 subunit were deleted with standard two-step PCR. All the subunits were cloned into the pCAG vector, with an amino-terminal Flag-tag and a carboxy-terminal His₁₀-tag at the α1 and β3 subunits. For western blotting to examine the stoichiometry of the α1 and β3 subunits, β3 subunit was cloned into the pCAG vector with amino-terminal twin strep-tag. HEK293F suspension cells (Thermo Fisher Scientific, R79007) were cultured in Freestyle 293 medium (Thermo Fisher Scientific) at 37 °C, supplied with 5% CO₂ under 60% humidity. When cell density reached 1.5-2.0 × 10⁶ cells per mL, a mixture of expression plasmids including 0.75 mg α1, 0.6 mg α2δ−1 and 0.5 mg β3, and 3 mg polyethylenimine (Polysciences) were added into the cell culture for transient expression of human Ca_v2.3 complex.

Sf9 insect cells (Thermo Fisher Scientific, Waltham, MA, USA) were cultured in Sf-900 III serum-free medium (Thermo Fisher Scientific) or SIM SF serum-free medium (Sino Biological) at 27 °C. HEK293F suspension cells (Thermo Fisher Scientific) were cultured in FreeStyle 293 medium (Thermo Fisher Scientific) or SMM 293-TI medium (Sino Biological) supplemented with 1% fetal bovine serum (FBS) at 37 °C with 6% CO₂ and 70% humidity. AD293 adherent cells (Agilent Technologies) were cultured in Dulbecco’s Modified Eagle Medium (Gibco) supplemented with 10% FBS at 37 °C with 5% CO₂. The cell lines were routinely checked to be negative for mycoplasma contamination but have not been authenticated.

Sf9 insect cells (Thermo Fisher Scientific) were cultured in Sf-900 III serum-free medium (Gibco) or SIM SF serum-free medium (Sino Biological) at 27°C. HEK293F suspension cells (Thermo Fisher Scientific) were cultured in FreeStyle 293 medium (Thermo Fisher Scientific) supplemented with 1% fetal bovine serum at 37°C with 6% CO₂ and 70% humidity. The cell lines were routinely checked to be negative for mycoplasma contamination but have not been authenticated.

Viral proteins were produced in HEK293F suspension cells (ThermoFisher) and purified as previously described.⁸ (link) Recombinant SARS-CoV-2-6P-Mut7 S protein for nsEM was produced and purified as previously described.⁷¹ (link) His-tagged E2 was purified by affinity purification using Ni-NTA agarose beads followed by size-exclusion chromatography. Monoclonal antibodies were produced as previously described.⁸ (link) Briefly, after co-transfection of the HC and LC plasmids in a 1:1 ratio and harvest after 5 days, the filtered supernatant was run over 10 mL protein G columns (Pierce). After elution, the purified antibodies were buffer exchanged to PBS using 100kDa Vivaspin6 columns. The IgG concentration was measured on a NanoDrop One (Thermofisher), diluted with PBS to 1mg/mL and stored at 4°C before performing the cFAE protocol.

SP2/0 mouse myeloma cells were grown in RPMI 1640 medium (Gibco, Thermo Fisher, USA) supplemented with 10% fetal bovine serum (FBS; Gibco) at 37 °C. African green monkey kidney VeroE6 cells were cultured in DMEM (Gibco, USA) supplemented with 10% FBS. HEK 293 F suspension cells (Thermo Fisher) were grown in FreeStyle 293 expression medium (Gibco). ExpiCHO-S™ cells (Thermo Fisher) were grown in ExpiCHO expression medium (Gibco). SARS-CoV-2 clinical isolate nCoV-SH01 (GenBank: MT121215.1)⁴⁶ was expanded in VeroE6 cells and virus titers were expressed as plaque forming units (PFU) per mL. All the infection experiments were performed in the biosafety level-3 (BSL-3) laboratory of Fudan University.