Pseudovirus particles expressing spike proteins were produced as described previously with plasmids expressing either ancestral or delta spike proteins61, (link)
62 (link). To measure neutralizing capacity of plasma and BALF, HEK-293 cells transduced to express ACE-2 were seeded onto CellCarrier-384 Ultra Microplates, PDL coated (PerkinElmer, Vic), and incubated overnight. The next day, plasma or BALF was diluted in DMEM media (Life Technologies, Thermo Fisher Scientific, NSW) and incubated at 37 °C for 1 h in the presence of pseudovirus. After incubation, cells were spinoculated (1 h, 35 °C, 800 × g) with virus and antibody then left for 72 h at 37 °C. Cells were then fixed in freshly prepared 4% paraformaldehyde (Life Technologies, Thermo Fisher Scientific, NSW) followed by nuclear staining with DAPI (Life Technologies, Thermo Fisher Scientific, NSW). Cells were analyzed for GFP fluorescence using an Opera Phenix High-Content Screening System (PerkinElmer, Vic) and Harmony® high-content analysis software (Perkin Elmer, Vic) provided by the Sydney Cytometry Facility (Charles Perkins Centre, NSW).
62 (link). To measure neutralizing capacity of plasma and BALF, HEK-293 cells transduced to express ACE-2 were seeded onto CellCarrier-384 Ultra Microplates, PDL coated (PerkinElmer, Vic), and incubated overnight. The next day, plasma or BALF was diluted in DMEM media (Life Technologies, Thermo Fisher Scientific, NSW) and incubated at 37 °C for 1 h in the presence of pseudovirus. After incubation, cells were spinoculated (1 h, 35 °C, 800 × g) with virus and antibody then left for 72 h at 37 °C. Cells were then fixed in freshly prepared 4% paraformaldehyde (Life Technologies, Thermo Fisher Scientific, NSW) followed by nuclear staining with DAPI (Life Technologies, Thermo Fisher Scientific, NSW). Cells were analyzed for GFP fluorescence using an Opera Phenix High-Content Screening System (PerkinElmer, Vic) and Harmony® high-content analysis software (Perkin Elmer, Vic) provided by the Sydney Cytometry Facility (Charles Perkins Centre, NSW).
