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.
S1300exi flow cytometer
Manufactured by Stratedigm
The S1300Exi Flow Cytometer is a laboratory instrument that enables the analysis and sorting of cells or particles suspended in a fluid stream. It utilizes the principles of flow cytometry to detect and measure various characteristics of individual cells or particles as they pass through a laser beam.
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10 protocols using s1300exi flow cytometer
1
Quantifying SARS-CoV-2 Pseudovirus Infectivity
2
ACE2-RBD Binding Assay by Flow Cytometry
3
ACE-2 Receptor Binding Assay
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ACE-2 expressing 293T cells were incubated with 200 nM of RBD antigen in PBS for 1hr on ice. Cells were resuspended in 50μL of secondary stain containing streptavidin-PE (Invitrogen) at a 1:200 dilution and incubated for 30 min on ice. Cell binding was analyzed by flow cytometry using a Stratedigm S1300Exi Flow Cytometer equipped with a 96 well plate high throughput sampler. Rsulting data were analyzed using FlowJo (10.7.1).
4
Quantifying Spike Protein Expression
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To compare the relative surface expression of pseudovirus spike variant proteins, we plated 400,000 293T cells per well of a 12-well plate. 24 h later, 1 μg of variant spike expression plasmid and transfected using PEI. Cells were incubated for 48 h at 37°C and harvested into PBS+ cells transfected with each vector were divided and stained with 5 μg/mL of either S309, ADI-55689, or ADI-56046 for 30 minutes at room temperature. Cells were then washed with 1 mL PBS+, spun at 900 × g, and stained with 2 μg/mL of anti-human IgG-AF647 polyclonal antibody (Invitrogen) for 30 minutes at room temperature. Cells were washed with 1 mL of PBS+, spun at 900 × g, resuspended in 100 μL of PBS+, fixed with 100 μL 4% PFA and analyzed on a Stratedigm S1300Exi Flow Cytometer.
5
Quantifying Pseudotyped Lentiviral Infectious Units
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To determine the infectious units of pseudotyped lentiviral vectors, we plated 400,000 293T-ACE2 cells per well of a 12-well plate. 24 h later, three ten-fold serial dilutions of neat pseudovirus supernatant were made in 100 μL, which was then used to replace 100 μL of media on the plated cells. 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-expressing 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, each transduced ZsGreen cell was assumed to represent a single infectious unit.
6
Quantifying Lentiviral Pseudovirus Infectivity
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To determine the infectious units of pseudotyped lentiviral vectors, we plated 400,000 293T-ACE2 cells per well of a 12-well plate. 24 h later, three ten-fold serial dilutions of neat pseudovirus supernatant were made in 100 μL, which was then used to replace 100 μL of media on the plated cells. 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, each transduced ZsGreen+ cell was assumed to represent a single infectious unit.
7
Quantification of Pseudovirus Infectivity
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To determine the infectious units of pseudotyped lentiviral vectors, we plated 400,000 293T-ACE2 cells per well of a 12-well plate. 24 h later, three ten-fold serial dilutions of neat pseudovirus supernatant were made in 100 μL, which was then used to replace 100 μL of media on the plated cells. 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-expressing 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, each transduced ZsGreen cell was assumed to represent a single infectious unit.
8
Quantifying Pseudovirus Spike Variant Expression
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To compare the relative surface expression of pseudovirus spike variant proteins, we plated 400,000 293T cells per well of a 12-well plate. 24 h later, 1 μg of variant spike expression plasmid and transfected using PEI. Cells were incubated for 48 h at 37°C and harvested into PBS+ cells transfected with each vector were divided and stained with 5 μg/mL of either S309, ADI-55689, or ADI-56046 for 30 min at room temperature. Cells were then washed with 1 mL PBS+, spun at 900 x g, and stained with 2 μg/mL of anti-human IgG-AF647 polyclonal antibody (Invitrogen) for 30 min at room temperature. Cells were washed with 1 mL of PBS+, spun at 900 x g, resuspended in 100 μL of PBS+, fixed with 100 μL 4% PFA and analyzed on a Stratedigm S1300Exi Flow Cytometer.
9
Quantifying Pseudovirus Infectious Units
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To determine the infectious units of pseudotyped lentiviral vectors, we plated 400,000 293T-ACE2 cells per well of a 12-well plate. 24 h later, three ten-fold serial dilutions of neat pseudovirus supernatant were made in 100 μL, which was then used to replace 100 μL of media on the plated cells. 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-expressing 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, each transduced ZsGreen cell was assumed to represent a single infectious unit.
10
ACE2-RBD Binding Assay by Flow Cytometry
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ACE2 expressing 293T cells were a gift from Michael Farzan (Scripps Florida) and Nir Hacohen (Broad Institute) (6 ) and were incubated with 200 nM of RBD antigen in PBS for 1hr on ice. Cells were resuspended in 50μL of secondary stain containing streptavidin-PE (Invitrogen) at a 1:200 dilution and incubated for 30 min on ice. Cell binding was analyzed by flow cytometry using a Stratedigm S1300Exi Flow Cytometer equipped with a 96 well plate high throughput sampler. Resulting data were analyzed using FlowJo (10.7.1).
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