Mini bioreactor centrifuge tube

ExpiCHO cells were seeded into 50 ml Mini Bioreactor Centrifuge Tube (Corning) at 6 × 10⁶ cells/ml in 5 ml ExpiCHO Expression medium (Life technology). Plasmids encoding SARS-CoV-2 Wuhan, Alpha (B.1.1.7), Delta (B.1.617.2) or Omicron (B.1.1.529) Spike protein (5 mg) were diluted in OptiPRO SFM (Life Technology) and mixed with Expifectamine CHO Reagent (Life Technology). After 1 min incubation at room temperature, transfection mixes were added to cell suspensions. Next, cells were incubated at 37°C 8% CO₂ with an orbital shaking speed of 120 rpm for the following 48 hours.
Transiently transfected cells were harvested and washed with PBS, 1% BSA, 2 mM EDTA. Cells were counted, dispensed into round-bottom 96 well plates (Corning) and incubated with human IgG Fc-conjugated ACE2 serial dilutions (concentration range: 30’000 – 0.17 ng/ml) for 45 min at 4°C. After two washes, Alexa Fluor647 Goat Anti-Human IgG secondary Ab (1.5 mg/ml) (Jackson Immunoresearch) was added to the cells, which were then incubated for 30 min at 4°C in the dark. Cells were washed twice and resuspended in wash buffer for data acquisition at ZE5 cytometer (Biorad). To assess Spike protein expression level, an aliquot of each transfectant cell line was stained with 10 mg/ml of S2P6 antibody (Pinto et al., Science 2021) for 45 min at 4°C.

ExpiCHO cells were seeded into 50 ml Mini Bioreactor Centrifuge Tube (Corning) at 6 × 10⁶ cells per ml in 5 ml ExpiCHO Expression medium (Life technology). Plasmids encoding SARS-CoV-2 Wuhan, Alpha (B.1.1.7), Delta (B.1.617.2) or Omicron (B.1.1.529) spike protein (5 mg) were diluted in OptiPRO SFM (Life Technology) and mixed with Expifectamine CHO Reagent (Life Technology). After incubation for 1 min at room temperature, transfection mixes were added to the cell suspensions. Next, the cells were incubated at 37 °C under 8% CO₂ with an orbital shaking speed of 120 rpm for the next 48 h.
Transiently transfected cells were collected and washed with PBS, 1% BSA and 2 mM EDTA. Cells were counted, dispensed into round-bottom 96-well plates (Corning) and incubated with human IgG Fc-conjugated ACE2 serial dilutions (concentration range: 30,000–0.17 ng ml⁻¹) for 45 min at 4 °C. After two washes, Alexa Fluor647 Goat Anti-Human IgG secondary antibodies (1.5 mg ml⁻¹) (Jackson Immunoresearch) was added to the cells, which were then incubated for 30 min at 4 °C in the dark. The cells were washed twice and resuspended in wash buffer for data acquisition at ZE5 cytometer (Bio-Rad). To assess spike protein expression level, an aliquot of each transfectant cell line was stained with 10 mg ml⁻¹ of S2P6 antibody³⁵ for 45 min at 4 °C.