Genejammer

One day prior to transfection, 293 T cells were plated at a density of 0.5 × 10⁶ cells in a 6-well plate and transfected with 1 μg plasmid DNA using Gene Jammer (Agilent Technologies). Forty-eight hours post transfection, culture supernatants were collected, and cells were lysed using cell lysis buffer (Cell Signaling) containing protease inhibitor cocktail (Cell Signaling). Approximately 50 μg of culture supernatants and cell lysates were run with an Odyssey Protein Molecular weight ladder (Licor) on 4–12% pre-cast bis-tris gel (Invitrogen). The separated peptides were transferred to PVDF membrane (iblot 2, Thermo Fisher). The membrane was blocked with Odyssey blocking buffer (Licor) for 1 h at room temperature. Heavy and light chains were detected using IRDye 680RD goat anti-human IgG (H + L) (Licor). The blot was scanned using Odyssey CLx (Licor).

SARS-CoV-2 pseudovirus was produced using HEK293T cells transfected with a 1:1 ratio of IgE-SARS-CoV-2 S plasmid (Genscript) and pNL4-3.Luc.R-E⁻ plasmid (NIH AIDS reagent) using Gene Jammer (Agilent) as transfection reagent. Forty-eight hours post transfection, transfection supernatant was collected, enriched with fetal bovine serum (FBS) to 12% final volume, and stored at −80°C. SARS-Cov-2 pseudovirus neutralization assay was set up using D10 medium (DMEM supplemented with 10% FBS and 1× penicillin-streptomycin) in a 96-well format using huCHOAce2 cells (Creative Biolabs, catalog no. VCeL-Wyb019). For neutralization assay, 10,000 CHO-ACE2 cells were plated in 96-well plates and rested overnight at 37°C and 5% CO₂ for 24 h. CTB-ACE2 cells at specific weights were incubated with a fixed amount of SARS-Cov-2 pseudovirus for 90 min at room temperature in complete medium. After centrifugation at 15,005 rpm for 10 min, the supernatant of this mix was added to huCHOAce2 cells and allowed to incubate in a standard incubator (37% humidity, 5% CO₂) for 72 h. After 72 h, cells were lysed using the Britelite Plus luminescence reporter gene assay system (PerkinElmer, catalog no. 6066769), and relative light units were measured using a BioTek plate reader. Percent neutralization was calculated using GraphPad Prism 8.

Transfections were performed using Genejammer (Agilent Technologies, Cheshire, UK) or FuGENE (Promega, Southampton, UK) transfection reagents according to the manufacturer's instructions or the calcium phosphate precipitation procedure modified by the use of {N,N-bis(2-hydroxyetyhl)-2-aminoethanesulfonic acid}-buffered saline (pH 7.06) as previously described (Batchelor and O'Hare, 1992 (link)). Routinely, 0.5–1 μg of the appropriate expression vector was transfected with amounts of DNA normalized using pUC19 carrier DNA.