Alexa fluor 647 conjugated antibody

To determine the infectious units and infection rates of SARS-CoV-2 pseudovirus, we plated 400,000 293T-ACE2 cells per well of a 12-well plate. SARS-CoV-2 pseudovirus supernatants that were produced in parallel under identical conditions (as described above) were added 24 h later in three ten-fold serial dilutions. Cells were incubated for 48 h at 37°C/5% CO₂ to allow for expression of ZsGreen reporter gene and harvested with Trypsin-EDTA (Corning). Cells were resuspended in PBS supplemented with 2% FBS (PBS+), and analyzed on a Stratedigm S1300Exi Flow Cytometer to determine the percentage of ZsGreen⁺ cells. Infectious units were calculated by determining the percentage of infected cells in wells exhibiting linear decreases in transduction and multiplying by the average number of cells per well determined at the initiation of the assay. At low MOI (infection rates <10%), each transduced ZsGreen⁺ cell was assumed to represent a single infectious unit. Surface expression of ACE2 on 293T and 293T-ACE2 cells was confirmed by staining with 1 μL of anti-human ACE2, Alexa Fluor 647-conjugated antibody (R&D). Cells were incubated for 10 min at 25°C prior to running on a Stratedigm S1300Exi Flow Cytometer.